The orphan receptor steroidogenic factor-1 (SF-1) plays a major role in adrenal and gonadal development and sexual differentiation, and it has been shown to interact with shared promoter elements to increase the expression of cytochrome P450 side-chain cleavage (P450(SCC)) and other steroid hydroxylases in vitro. We took a step-wise approach to define the role of SF- 1 in regulation of hormone-induced steroidogenesis. In a mouse Leydig cell line (MA-10) we show that hCG and forskolin are effective inducers of progesterone production and P450(SCC) expression. In contrast, endogenous SF- 1 expression was not increased by either hCG or forskolin. Similarly, these agents did not enhance the activity of SF-1 promoter transfected into MA-10 cells. The transcriptional activity of SF-1, measured by induction of an SF- 1 synthetic reporter, was only minimally increased by forskolin. Within the context of the rat P450(SCC) promoter, mutation of the two SF-1-binding sites caused a dramatic decrease in constitutive activity of this promoter, but the degree of induction by 8-bromo-cAMP was only reduced from 7.9-fold to 5.9- fold. We conclude that SF-1 is required for the constitutive activity of P450(SCC), but that it does not play a direct role in the early induction of steroidogenesis by hCG or forskolin in MA-10 cells.