Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in LBNF₁ rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for two weeks; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 weeks; testosterone also reduced the TDI demonstrating that testosterone had a definite inhibitory effect, independent of pituitary hormones. Further more, in this protocol we showed that TDI in the hypophysectomized-testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRH-antagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 days in untreated, GnRH-ant plus flutamide treated, or GnRH-ant plus testosterone treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus T treated rats. We conclude that both testosterone and FSH indiudually inhibit spermatogonial differentiation after irradiation but testosterone is a more highly potent inhibitor than is FSH.