The effects of dimethylsulfoxide (DMSO) on steroidogenesis in MA-10 mouse Leydig tumor cells and R2C rat Leydig tumor cells were compared. MA-10 cells produce low basal levels of progesterone (as the major steroid synthesized), which increase several hundred-fold when stimulated with tropic hormone or the cAMP analog (Bu)2cAMP. R2C cells, on the other hand, constitutively produce high levels of progesterone in the absence of hormone stimulation of any kind. When incubated in the presence of 5% DMSO, MA-10 cells demonstrated an almost complete inhibition of progesterone production, whereas the synthesis of this steroid was virtually unaffected in R2C cells. The inhibition of steroid production in the MA-10 cell could not be attributed to an effect on protein synthesis, because this was unaffected by DMSO during the course of the incubations. The activity of the cholesterol side-chain cleavage enzyme was also unaffected by DMSO, as demonstrated by incubation of the cells with 22R-hydroxycholesterol. The production of cAMP in response to tropic hormone (hCG) and forskolin stimulation was significantly inhibited in MA-10 cells, but was much less affected in R2C cells in response to forskolin treatment. However, DMSO appeared to have no effect on the overall phosphorylation of proteins when tested either in a completely in vitro system or when MA-10 cell homogenates were used as a source of exogenous protein. Strikingly, DMSO treatment resulted in the highly specific inhibition of a series of 30-kilodalton mitochondrial proteins (named StAR for Steroidogenic Acute Regulatory protein), which we have recently shown to be indispensable for the production of steroids in MA-10 cells. The synthesis of these same proteins was much less affected in R2C cells. Although the mechanism of action by which DMSO inhibits steroidogenesis remains unknown, these results show that its action results in the complete cessation of synthesis of the StAR protein, which is required for the acute regulation of steroidogenesis in MA-10 cells.