Secretion of Müllerian-inhibiting substance (MIS) by Sertoli cells of the fetal testis and subsequent regression of the Müllerian ducts in the male embryo is a crucial event that contributes to proper sex differentiation. The zinc finger transcription factor GATA-4 and nuclear receptor SF-1 are early markers of Sertoli cells that have been shown to regulate MIS transcription. The fact that the GATA and SF-1 binding sites are adjacent to one another in the MIS promoter raised the possibility that both factors might transcriptionally cooperate to regulate MIS expression. Indeed, coexpression of both factors resulted in a strong synergistic activation of the MIS promoter. GATA-4/SF-1 synergism was the result of a direct protein-protein interaction mediated through the zinc finger region of GATA-4. Remarkably, synergy between GATA-4 and SF-1 on a variety of different SF-1 targets did not absolutely require GATA binding to DNA. Moreover, synergy with SF-1 was also observed with other GATA family members. Thus, these data not only provide a clearer understanding of the molecular mechanisms that control the sex-specific expression of the MIS gene but also reveal a potentially novel mechanism for the regulation of SF-1-dependent genes in tissues where SF-1 and GATA factors are coexpressed.