The scavenger receptor, class B, type I (SR-BI), is the predominant receptor that supplies plasma cholesterol to steroidogenic tissues in rodents. We showed previously that steroidogenic factor-1 (SF-1) binds a sequence in the human SR-BI promoter whose integrity is required for high-level SR-BI expression in cultured adrenocortical tumor cells. We now provide in vivo evidence that SF-1 regulates SR-BI. During mouse embryogenesis, SR-BI mRNA was initially expressed in the genital ridge of both sexes and persisted in the developing testes but not ovary. This sexually dimorphic expression profile of SR-BI expression in the gonads mirrors that of SF-1. No SR-BI mRNA was detected in the gonadal ridge of day 11.5 SF-1 knockout embryos. Both SR-BI and SF-1 mRNA were expressed in the cortical cells of the nascent adrenal glands. These studies directly support SF-1 participating in the regulation of SR-BI in vivo. We examined the effect of cAMP on SR-BI mRNA and protein in mouse adrenocortical (Y1-BS1) and testicular carcinoma Leydig (MA-10) cells. The time courses of induction were strikingly similar to those described for other cAMP- and SF-1-regulated genes. Addition of lipoproteins reduced SR-BI expression in Y1-BS1 cells, an effect that was reversed by administration of cAMP analogs. SR-BI mRNA and protein were expressed at high levels in the adrenal glands of knockout mice lacking the steroidogenic acute regulatory protein; these mice have extensive lipid deposits in the adrenocortical cells and high circulating levels of ACTH. Taken together, these studies suggest that trophic hormones can override the suppressive effect of cholesterol on SR-BI expression, thus ensuring that steroidogenesis is maintained during stress.