Previously we examined that inhibin-α subunit, transforming growth factor-β₁ (TGF-β₁) and epidermal growth factor (EGF) were expressed in sex-, cell- and stage-specific manners in perinatal rat gonads. To clarify effects of these growth factors on the rat gonadal differentiation and development, indifferent gonadal primordia with mesonephric tubules on gestational day 13 were cultured in vitro for 4 days in serum-free CMRL-1066 medium with inhibin, TGF-β₁, EGF, anti-sera against these growth factors, testosterone or estradiol-17β, and then morphologically examined with reference to seminiferous tubule formation, germ cell division, Wolffian and Müllerian duct development. In male gonads, anti-inhibin-α serum suppressed the seminiferous tubule formation but inhibin, TGF-β₁, EGF or steroid hormones did not affect on the tubule formation, germ cell proliferation nor gonoduct development. Seminiferous tubules in male gonads cultured in the medium containing anti-inhibin-α serum were incomplete and irregular in shape. On the other hand, in female gonads, inhibin suppressed the germ cell division and anti-inhibin-α serum led to the necrosis of germ cells, but other factors affected to neither sex cord formation nor germ cell division. Testosterone and estradiol-17β stimulated female Wolffian and Müllerian duct development, respectively. These results indicate that inhibin induces the seminiferous tubule formation and suppresses the female germ cell division in developing rat gonads in vitro.