Studio 54 was originally built as the Gallo Opera House. The theatre opened in November 1927 with a production of La Boheme, San Carlo Opera Company's first production of an unsuccessful three-week season.

After financial difficulty with the opera, impresario Fortune Gallo soon turned to producing legitimate shows such as Electra, featuring Antoinette Perry (for whom the Tony Awards are named), and the Oscar Hammerstein musical Rainbow.

After continued failure, the theatre gained a reputation for bad luck and was sold in foreclosure shortly after the stock market crash of 1929.

In 1930 the space was renamed the New Yorker Theatre and housed productions like Ibsen's The Vikings. Over the next few years, the theatre continued to house flops. The space changed hands several times and became the Casino de Paris, the Palladium, and the Federal Music Theatre restored and run by the WPA.

The space was purchased by CBS in 1942 and became the CBS Radio Playhouse Number 4, and later Studio 52. Over the next three decades it was home to radio broadcasts and, later, renowned television shows like The Jack Paar Show, The Jack Benny Show, The Johnny Carson Show and such varied fare as The $64,000 Question, Beat the Clock and Captain Kangaroo.

In the late 1970s, the space was sold and transformed into the nightclub Studio 54, one of the most famed discos of all time. After the club was closed in 1986, it became a venue for rock concerts in the late '80's. Thereafter, the space was largely neglected until 1998 when Roundabout discovered the theatre, a jewel in the rough on the northern-most edge of the Broadway district. Today, Studio 54 is a permanent home for Roundabout Theatre Company and will house their musicals and large-scale productions for years to come.

Productions by Roundabout Theatre include their revival of Cabaret, Assassins, Pacific Overtures and A Streetcar Named Desire.