The present studies examined the effects of hCG on steroidogenic enzyme activities involved in the metabolism of testosterone or dihydroststosterone in cultured rat neonatal interstitial cells. 5α-reductase and 17β-hydroxysteroid dehydrogenase activities, which are involved in the conversion of testosterone to dihydrotestosterone and androstenedione, respectively, were low in cultured neonatal interstitial cells, were unresponsive to hCG and declined to undetectable levels during 14 days of culture. However, Δ5-3β-hydroxysteroid dehydrogenase-isomerase activity, which is involved in the biosynthesis of testosterone, and 5α-androstane-3α-hydroxysteroid dehydrogenase and 5α-androstane-3β-hydroxysteroid dehydrogenase activities, which are involved in the conversion of dihydrotestosterone to 5α-androstan-3α, 17β-diol and 5α-androstan-3β, 17β-diol, respectively, were maintained or increased by hCG during the same culture period. These results demonstrate that 5α-reductase and 17β-hydroxysteroid dehydrogenase activities do not play a significant role in regulating testosterone accumulation in fetal/neonatal Leydig cells, and they suggest that these cells are adapted to maintain high testosterone but low dihydrotestosterone levels. The present results demonstrate also that fetal/neonatal Leydig cells differ from immature or adult Leydig cells with respect to the sensitivity of 5α-reductase activity to LH/hCG.