Irradiation of LBNF₁ rat testes induces arrest of spermatogonial differentiation, which can be reversed by suppression of testosterone with GnRH antagonist treatment. The cause of the arrest is unknown. We investigated the time course and hormonal effects on radiation-induced arrest and changes in interstitial fluid volume. We postulated that the edema evident in irradiated testes caused the differentiation blockade. Rat testes were irradiated with 3.5 or 6 Gy. Interstitial fluid testosterone (IFT) increased between 2 and 6 weeks after irradiation, followed by increased interstitial fluid volume at 6 weeks and spermatogonial blockade at 8 weeks. Additional rats irradiated with 6 Gy were given GnRH antagonist, alone or with exogenous testosterone, for 8 weeks starting at 15 weeks after irradiation. In rats treated with GnRH antagonist, IFT started falling within 1 week of treatment, followed by interstitial fluid volume decreases at weeks 2 and 3, with recovery of spermatogenesis starting at week 4. Addition of exogenous testosterone largely blocked the effects of GnRH antagonist on IFT, interstitial fluid volume, and spermatogenesis. Thus the testicular edema was largely modulated by intratesticular testosterone levels. The time course of changes in the spermatogonial blockade more closely followed that of the testicular edema than of IFT, indicating that testosterone may block spermatogonial differentiation indirectly by producing edema. This conclusion was further supported by an experiment in which irradiated rats were treated with GnRH antagonist plus estrogen: the treatment further reduced IFT, interstitial fluid volume and reduced the time to initiation of recovery of spermatogonial differentiation. These results suggest that studies of the edematous process or composition of the fluid would help elucidate the mechanism of spermatogonial arrest in toxicant-treated rats.