We identified a regulatory region of the mouse CD4 promoter by both in vivo and in vitro analysis. The results of transient transfection assays indicated that the dominant transcription activating element within the CD4 promoter is located at -82 to -42. Elimination of this element, by linear deletion or specific mutation, significantly reduced transcriptional activity from this promoter. DNase I footprinting and gel mobility shift assays confirmed that the region -90 to -64 acts as the binding site of a specific nuclear factor, designated NF-CD4. In this region, an 11 bp core motif (CAACAACTGGG; -82 to -72) was found to be sufficient for the binding and transcriptional activation of the NF-CD4. This motif contains a consensus sequence for binding of c-Myb and proteins with helix-loop-helix structures. Indeed, bacterially-synthesized c-Myb specifically binds to this motif for NF-CD4. Furthermore, binding of NF-CD4 to the promoter region was specifically inhibited by the addition of anti-Myb antibodies. The evidence strongly suggests that c-Myb binds, in a sequence-specific fashion, to the core region of the CD4 promoter defined by functional assays and that this proto-oncogene product appears to play a role in the complex regulation of CD4 gene expression during T cell development.