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 while microsomal membrane cytochrome b₅ is undetectable. Outer mitochondrial membrane cytochrome b₅ present in the testis was localized in Leydig cells with cytochrome P-450₁₇ which catalyzes androgen genesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b₅ in rat testis microsomes 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-450₁₇-catalyzed reactions, 17-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b₅; preferential elevation of the 17-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 17-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b₅ not microsomal membrane cytochrome b₅ functions as an activator for androgengenesis in rat Laydig cells.