Previous studies from this laboratory have demonstrated the presence of two populations of rat Leydig cells (I and II), which differ in their capacity for hCG- or cAMP-stimulated testosterone production. In the present study, we examined the metabolism of 25-hydroxycholesterol in primary cultures of both populations of Leydig cells. 25-Hydroxycholesterol bypasses the cAMP-dependent transport mechanism to the mitochondrial cytochrome P-450 side-chain cleavage enzyme (P-450scc) required by cholesterol and thus provides an index of the relative activity of P-450scc. Incubation of Leydig cells with increasing concentrations of 25-hydroxycholesterol resulted in a concentration-dependent increase in the amount of testosterone produced, with maximal amounts in both populations being reached at 25-hydroxycholesterol concentrations of 5 microM or greater. Population II produced more than twice as much testosterone as population I Leydig cells, whether incubated with 25-hydroxycholesterol or with 8-bromo-cAMP. Each population of Leydig cells produced 2.5-fold greater amounts of testosterone when incubated with 25-hydroxycholesterol than when incubated with 8-bromo-cAMP. In both populations of Leydig cells, cAMP-stimulated testosterone production was not different in cells cultured for 24 h from that in freshly isolated Leydig cells. These data suggest that cholesterol transport to P-450scc limits maximal testosterone production, and that the difference in hCG- or cAMP-stimulated testosterone production between the two populations of Leydig cells is primarily due to differences in P-450scc activity between the two populations and is not a result of population I consisting mostly of damaged Leydig cells.