The relationship of maximal testosterone production to the amounts of cholesterol side-chain cleavage (P450scc), 17 alpha-hydroxylase/C17-20 lyase (P450(17) alpha), and iron sulfur protein (ISP) reductase was determined in Leydig cells from four inbred strains of mice (RF/J, SWR/J, C3H/He, and DBA/2). The amounts of P450scc, P450(17) alpha, and ISP reductase were also determined in adrenal glands of the same mice. cAMP-stimulated testosterone production and P450scc protein were high in RF/J and SWR/J compared to C3H/He and DBA/2 Leydig cells. A significant correlation between the amount of this enzyme and the capacity for testosterone production was found (r = 0.89; P less than 0.0005). ISP reductase was highest in RF/J, SWR/J, and C3H/He Leydig cells, which are significantly different from DBA/2. No significant differences in the amount of P450(17) alpha in Leydig cells from the four strains could be detected, and neither ISP reductase nor P450(17) alpha correlated with testosterone production. To ascertain if tissue-specific factors affect the expression of these enzymes, P450scc, ISP reductase, and P450(17) alpha were quantitated in adrenals from the same mice. P450scc and ISP reductase were expressed differently in adrenals compared to Leydig cells; levels of both proteins were high in C3H/He and RF/J adrenals compared to SWR/J and DBA/2. P450scc and ISP reductase were coordinately expressed in the adrenal, unlike in Leydig cells. P450(17) alpha was not detected in mouse adrenal glands. The results of this study suggest that strain-related differences in the capacity of Leydig cells for testosterone production may be determined by the amount of P450scc per Leydig cell. The expression of P450scc and ISP reductase in Leydig cells and adrenal glands appears to be influenced by both genetic and tissue-specific factors.