Previous work established biochemical homology between proteins named SCc1 and SCc2 in FSH-treated Sertoli cell cultures and a family of 30-kDa proteins, collectively referred to as Steroidogenic Acute Regulatory protein (StAR), in hormone-treated adrenal and gonadal cells. The purpose here was to establish the presence of StAR in FSH-treated Sertoli cell cultures using a StAR-specific antiserum. Immunofluorescence microscopy and immunoblot analysis were used to detect StAR in Sertoli cell cultures and in R2C rat Leydig tumor cell cultures that served as a positive control for StAR expression. FSH dramatically enhanced the appearance of StAR in mitochondria of Sertoli cells, whereas R2C cells expressed StAR constitutively. It has been proposed that StAR participates in steroidogenesis by facilitating the conversion of cholesterol to pregnenolone by the cytochrome P450 side-chain cleavage enzyme. FSH-treated Sertoli cells were shown here to lack detectable levels of side-chain cleavage enzyme protein and RNA, while R2C cells expressed both. We conclude that the appearance of StAR in Sertoli cell mitochondria is regulated by FSH, but that its function in Sertoli cells is not associated with the conversion of cholesterol to pregnenolone.