Although GH plays a key role in postnatal growth and differentiation, its role in fetal differentiation is not clear at the present. The aim of the present study was to investigate whether GH plays a role in fetal sexual differentiation, and we used in vitro organ culture assay of sexual differentiation to determine this. The results showed that anti-rGH antibody blocked Wolffian duct differentiation specifically in the presence of fetal testes. Exogenous GH supplemented in the above experiment reversed the blocking effect of anti-GH. Among the other related products, insulin-like growth factor I was highly effective in reversing the anti-GH effect, insulin-like growth factor II was partially effective, but PRL was unable to reverse the anti-GH effect. GH itself was found to produce some masculinizing effect, as demonstrated by its ability to stabilize the Wolffian duct in female fetuses. The role of GH was further demonstrated by the observation that GH-immunoreactive material of the size of authentic GH was detected in the 18-day fetal reproductive tract, and the concentration of this material increased in response to progression of sexual differentiation. Determination of androgen-binding activity using Scatchard analysis on the cells isolated from the 18-day fetal reproductive tract indicated that androgen-binding activity increased after GH treatment of the cells. Thus, it may be concluded that GH influences male sexual differentiation and alters the androgen-binding activity of the fetal reproductive tract.