Expression of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is required for efficient and complete presentation of antigens by MHC class II molecules and a normal immune response. The Ii gene is generally co-regulated with the MHC class II molecules at the level of transcription and a shared SXY promoter element has been described. This report defines the proximal promoter region of Ii which may regulate Ii transcription distinct from MHC class II. In vivo genomic footprinting identified an occupied, imperfect CCAAT box and an adjacent GC box in the proximal region. These sites are bound in Ii-ositive cell lines and upon interferon-gamma induction of Ii transcription. In contrast, both sites are unoccupied in Ii-egative cell lines and in inducible cell lines prior to interferon-gamma treatment. Together these two sites synergize to stimulate transcription. Independently, the transcription factor NF-Y binds poorly to the imperfect CCAAT box with a rapid off rate, while Sp1 binds to the GC box. Stabilization of NF-Y binding occurs upon Sp1 binding to DNA. In addition, the half-life of Sp1 binding also increased in the presence of NF-Y binding. These findings suggest a mechanism for the complete functional synergy of the GC and CCAAT elements observed in Ii transcription. Furthermore, this report defines a CCAAT box of imperfect sequence which binds NF-Y and activates transcription only when stabilized by an adjacent factor, Sp1.