EGF is known to modulate a variety of cellular functions including differentiation. The aim of this investigation was to determine the role of EGF in androgen-induced masculine differentiation. Accordingly, a series of experiments were designed and the results are summarized as described below. (1) We found that the specific deprivation of EGF using anti-EGF serum during the period of masculine differentiation in an organ culture bioassay system resulted in the disintegration of the Wolffian system in a dose-dependent manner. (2) Exogenous EGF supplemented in the above experiment corrected the anti-EGF effect, suggesting a specific role of EGF. (3) Anti-EGF serum was also found to disrupt the differentiation even in the presence of exogenous testosterone, suggesting an effect independent of testosterone synthesis. (4) EGF was found to have a direct masculinizing effect both in vivo and in vitro; however, it was not able to mimic all masculinizing effects of testosterone. The mesonephric segment of the Wolffian duct was retained by EGF in the female fetal tract under in vitro conditions, and under in vivo conditions EGF was able to increase anogenital distance and to induce epididymis in some female fetal mice. (5) We were able to detect an EGF-like material in the fetal genital tract during differentiation and found that the level of this material increased with advancement of differentiation. Thus, it appears from the above results that EGF plays a role in testosterone-induced reproductive tract differentiation.