Depiction in the series
Season oneThe castle is first introduced in the second part of the season one premiere, mentioned in the book The Elements of Harmony: A Reference Guide. It specifies that the last known location of the Elements of Harmony is in the ancient castle of the royal pony sisters, and that the castle itself is located in what is now known as the Everfree Forest. Twilight Sparkle and her friends travel to the castle in order to retrieve the Elements and defeat Nightmare Moon.
In this episode, only two rooms in the castle are featured: the foyer, where the Elements of Harmony are found, and the throne room, where Twilight and her friends defeat Nightmare Moon.
Season fourIn the season four premiere, Princess Twilight Sparkle - Part 1, Twilight views a flashback in which the castle is in its prime. Unlike in Friendship is Magic, part 2, the throne room is shown to have two thrones instead of one. Within the flashback, Twilight witnesses Princess Luna destroy parts of the throne room and her transformation into Nightmare Moon.
In Princess Twilight Sparkle - Part 2, Nightmare Moon attacks Princess Celestia, destroying more of the throne room and much of the castle's surrounding architecture in the process. The Elements of Harmony are also shown, when Princess Celestia raises them up from a chamber underneath the throne room floor.
The castle and its inner workings are featured in greater detail in Castle Mane-ia when Twilight and her friends all find their way to the old castle for varying reasons. The first new room in the castle to be shown is the library, which houses an abundance of ancient books and a large stone Alicorn bust. Star-spiders have also taken up residence in the library.
The castle is shown to be filled with numerous secret doors and traps, some of which Twilight's friends inadvertently set off and get themselves trapped in and most of which are controlled by a large pipe organ in the castle basement. Other rooms and areas in the castle include the "Hall of Hooves"—a hallway lined with torch wall-mounts shaped like pony legs—a corridor lined with suits of pony armor, and a basement chamber that houses the aforementioned "Organ to the Outside". External shots of the castle show dragon statues on its ledges.One significant room discovered by Twilight, with the assistance of Spike and Angel, is a personal reading room for the royal sisters off the main library behind a secret door. This small chamber has a small reading couch and some throw pillows. The room also features a raised reading stand upon which rests the personal journal of Princess Celestia and Princess Luna from the time before Luna's fall to become Nightmare Moon. Illumination to the room is through two intact stained glass windows, a blue one depicting Luna's moon and a yellow one depicting Celestia's sun.
Despite having evidently been abandoned after Nightmare Moon's defeat, the castle remains in surprisingly good condition. The books in the library, despite being over a thousand years old, all appear in a usable condition, the various traps and pitfalls work smoothly, and the Organ to the Outside is in good working order and remains in tune. Even a small trapdoor that delivers food to Luna and Celestia's personal reading room appears to somehow still have access to fresh produce.
In the time following the castle's abandonment, it has also become the subject of Equestrian urban legends, such as the "Pony of Shadows", an ethereal being formed from remnants of Nightmare Moon's magic.In Power Ponies, Twilight and her friends are seen tidying the castle up.
Spike retrieves the Inspiration Manifestation spell from a hidden trapped chamber in the library of this castle in the episode of the same name.Twilight and her friends are seen in the castle again in Twilight's Kingdom - Part 1 in order to figure how to unlock the Tree of Harmony chest.
The castle appears in Princess Luna's dream in Do Princesses Dream of Magic Sheep? when she dreams of becoming Nightmare Moon again.
In The Cutie Re-Mark - Part 2, the castle appears in an alternate timeline in pristine condition, serving as Nightmare Moon's castle home. The castle's design in the episode is partially based on artwork shown both in The Art of Equestria and in a background image featured on Hubworld.com.
Depiction in books
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The castle appears in Friendship is Magic, as in the second of the two episodes adapted; is featured in Rainbow Dash and the Daring Do Double Dare, in which the Dual Stronghold is code for the Castle of the Two Sisters; serves as a secret meeting point of the Mane 6 in Rarity and the Curious Case of Charity; and is detailed in The Journal of the Two Sisters, including the construction of the Castle of the Two Sisters.
In the IDW comics' Reflections story arc, the Castle of the Two Sisters still stands in an alternate universe of Equestria. In FIENDship is Magic Issue 1, the original Castle of Two Sisters is shown visited by Radiant Hope in the past on page 21 and is mentioned on page 22. In Friends Forever Issue 16, the castle is visited by the Cutie Mark Crusaders. In My Little Pony: Friends Forever Issue #35 Twilight and Starlight Glimmer travel to the castle to "tidy up the library" only to end up in a competition.