CIITA is the mediator of MHC class II gene induction by interferon-γ (IFNγ). The CIITA gene is itself selectively activated via one of its four promoters (PIV). We show here that three cis-acting elements, the GAS, the E box, and the IRF-1–binding site, as well as the trans-acting factors Stat1 and IRF-1, are essential for activation of CIITA promoter IV by IFNγ. Stat1 binds to the GAS site only in the presence of the ubiquitous factor USF-1, which binds to the adjacent E box. Indeed, Stat1 and USF-1 bind to the GAS/E box motif in a cooperative manner. The specificity for CIITA activation by IFNγ is thus dictated by the GAS/E box motif and by the selective interaction of IFNγ-activated Stat1 and USF-1. This clarifies the missing link in the overall pathway of IFNγ activation of MHC-II expression.