Cytochrome P450 17α-hydroxylase/17–20 lyase (P450_C17) is a critical branchpoint enzyme for steroid hormone biosynthesis. During human gestation, P450_C17 is required for the production of dehydroepiandrostenedione sulfate by the fetal adrenal cortex and for testicular production of androgens that mediate male sexual differentiation. In this study, we investigate the regulation of the human CYP17 gene by two orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX1. In human embryos, SF-1 and DAX1 are expressed throughout the developing adrenal cortex from its inception at 33 days post conception (dpc). In contrast, P450_C17 expression, which commences between 41 and 44 dpc, is limited to the fetal zone. The 5′-flanking region of the human CYP17 gene contains three functional SF-1 elements that collectively mediate a ≥25-fold induction of promoter activity by SF-1. In constructs containing all three functional SF-1 elements, DAX1 inhibited this activation by ≥55%. In the presence of only one or two SF-1 elements, DAX1 inhibition was lost even though SF-1 transactivation persisted. These data suggest that efficient repression of SF-1-mediated activation of the human CYP17 gene by DAX1 requires multiple SF-1 elements. Opposing effects of SF-1 and DAX1 may fine tune the differential responses of various SF-1 target genes in different endocrine tissues.