Outer mitochondrial membrane cytochrome b5 is an isoform of microsomal membrane cytochrome b5. In rat testes the outer mitochondrial membrane cytochrome b5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b5 is undetectable. Outer mitochondrial membrane cytochrome b5 present in the testis was localized in Leydig cells with cytochrome P-45017alpha, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b5 stimulated the cytochrome P-45017alpha-catalyzed reactions, 17alpha-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b5 as follows: preferential elevation of the 17alpha-hydroxylase activity by outer mitochondrial membrane cytochrome b5 in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b5 at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b5 elevated primarily the production of 17alpha-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b5 and not microsomal membrane cytochrome b5 functions as an activator for androgenesis in rat Leydig cells.