Presence of identical mitochondrial proteins in unstimulated constitutive steroid-producing R2C rat Leydig tumor and stimulated nonconstitutive steroid-producing MA-10 mouse Leydig tumor cells. The acute regulation of steroidogenesis in steroidogenic tissues requires de novo protein synthesis. It is believed that these newly synthesized proteins are instrumental in the delivery of the substrate, cholesterol, to the inner mitochondrial membrane where the cholesterol side-chain cleavage complex converts cholesterol to pregnenolone. A number of studies have attempted to characterize the protein(s) synthesized in response to hormone stimulation and, hence, function in the delivery of cholesterol to the cholesterol side-chain cleavage complex. While a number of potential protein candidates have been described, there is at present no unequivocal evidence which indicates that they are involved in steroidogenic regulation. We and others have described proteins that are induced in a variety of steroidogenic tissues in response to hormone stimulation and are localized in the mitochondria of these tissues. In an attempt to determine whether these induced proteins may be involved in steroidogenesis, we compared mitochondrial protein profiles in two separate Leydig tumor cell line. One cell line, the MA-10 mouse Leydig tumor cell line, has a very low basal steroid production, but synthesizes large amounts of progesterone in response to both tropic hormone and cAMP analog. The other cell line, the R2C rat Leydig tumor cell line, produces constitutively large amounts of progesterone, which cannot be increased further with hormone stimulation. Two-dimensional polyacrylamide gel electrophoresis profiles of newly synthesized mitochondrial proteins demonstrated that four 30-kDa proteins are induced in response to hormonal stimulation in MA-10 cells. Further, it was shown that proteins identical to these induced proteins are present constitutively in the mitochondria of R2C cells and cannot be further increased with hormone stimulation. These results strongly suggest that the 30-kDa mitochondrial proteins shown to be induced in several steroidogenic tissues are involved in the acute regulation of steroid production. 