The forkhead protein A1 (FoxA1) is critical for the androgenic regulation of prostate-specific promoters. Prostate tissue rescued from FoxA1 knockout mice exhibits abnormal prostate development, typified by the absence of expression of differentiation markers and inability to engage in secretion. Chromatin immunoprecipitation and coimmunoprecipitation studies revealed that FoxA1 is one of the earliest transcription factors that binds to prostate-specific promoters, and that a direct protein-protein interaction occurs between FoxA1 and androgen receptor. Interestingly, evidence of the interaction of FoxA1 with other transcription factors is lacking. The upstream stimulatory factor 2 (USF2), an E-box-binding transcription factor of the basic-helix-loop-helix-leucine-zipper family, binds to a consensus DNA sequence similar to FoxA1. Our in vitro and in vivo studies demonstrate the binding of USF2 to prostate-specific gene promoters including the probasin promoter, spermine-binding protein promoter, and prostate-specific antigen core enhancer. Furthermore, we show a direct physical interaction between FoxA1 and USF2 through the use of immunoprecipitation and glutathione-S-transferase pull-down assays. This interaction is mediated via the forkhead DNA-binding domain of FoxA1 and the DNA-binding domain of USF2. In summary, these data indicate that USF2 is one of the components of the FoxA1/androgen receptor transcriptional protein complex that contributes to the expression of androgen-regulated and prostate-specific genes.