Upon stimulation of Leydig cells with luteinizing hormone (LH) or dibutyryl-3',5'-cyclic AMP (Bt₂cAMP) at 37°C, two mitochondrial phosphoproteins accumulate with the same stimulant dose response as the increased rate of testosterone synthesis. The proteins pp32 and pp30 have apparent isoelectric points of 6.6 and 6.5 and molecular weights of approximately 32 and 30 kDa respectively, as determined by two-dimensional polyacrylamide gel electrophoresis. These two phosphoproteins are not detected in mouse adipose or liver cells nor in the total testicular cell population, of which Leydig cells constitute a small percentage. However, both proteins are also observed in mouse adrenal cells stimulated by ACTH or Bt₂cAMP. The appearance of pp32 and pp30 is prevented by inhibitors of cytosolic protein translation, indicating that only newly synthesized protein is available as a substrate for phosphorylation. Proteolytic peptide mapping indicates that both of these mouse Leydig and adrenal proteins have structural similarity to pp30 (formerly denoted as i_b), the 30 kDa mitochondrial phosphoprotein that we have observed previously in peptide hormone or Bt₂cAMP-stimulated rat adrenal cortex (Pon, L.A, Hartigan, J.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 13309–13316; Alberta, J.A., Epstein, L.F., Pon, L.A. and Orme-Johnson, N.R. (1989) J. Biol. Chem. 264, 2368–2372) and rat corpus luteum cells (Pon, L.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6694–6599). Since pp32 is a larger mitochondrial protein of similar primary structure to pp30, it is a potential precursor of this protein. Finally, the detection of the mitochondrial phosphoprotein pp30 in a third steroidogenic tissue type and a third species provides further correlative evidence that the production of pp30 may be an integral part of the subcellular mechanism by which peptide hormones stimulate steroid hormone biosynthesis.