Müllerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (-6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I.