Leydig cells from 40 days old rats were incubated with or without human growth hormone (hGH) or insulin-like growth factor I (IGF-I) in the presence or absence of human chorionic gonadotropin (hCG), and testosterone and cyclic AMP (cAMP) levels in the medium were measured. Neither hGH nor IGF-I increased testosterone production in the absence of hCG in concentrations up to 1000 and 100 ng/ml, respectively. However, both peptides increased hCG-induced testosterone production in a dose-dependent manner. The maximal stimulatory concentrations of hGH and IGF-I were 100 and 50 ng/ml, respectively. Human GH did not further enhance the IGF-I-stimulated steroidogenesis. The hGH-augmented steroidogenesis was inhibited by anti-hGH IgG and anti-IGF-I IgG. hGH also enhanced hCG-stimulated cAMP production time dependently, suggesting that the stimulatory effect of hGH on steroidogenesis was due to an increased cAMP production. These data suggest that the effect of hGH might be mediated by locally produced IGF-I, which may act as a modulator on gonadal development in the presence of gonadotropin.