It is known that steroid hormones can affect neuronal susceptibility to different types of insults, including oxidative stress. Using an in vitro/ex vivo model, we have previously shown that cerebellar granule cells prepared from neonatal rats treated with a single dose of testosterone are less vulnerable to oxidative stress-induced cell death, via a mechanism involving an upregulation of the cellular antioxidant defenses. Whether the testosterone protective action on cerebellar granule cells was direct or indirect remained to be clarified. Therefore, in this study we have investigated the effects of in vitro testosterone treatment, to see whether it also protects cerebellar granule cells from oxidative stress-induced damage. Cerebellar granule cells treated with 10(-6) M testosterone for 48 h were found less susceptible to damage induced by 50 microM hydrogen peroxide, as shown by a 30% decrease in the number of cells with apoptotic morphology. The addition of the androgen receptor antagonist flutamide abolished the protective effect of testosterone, suggesting an androgen receptor-mediated mechanism. This hypothesis was further supported by the presence of the androgen receptor in cultured cerebellar granule cells. The activity of the antioxidant enzyme catalase was also measured, and a 2-fold increase was detected in the testosterone treated cells, but not in the cells co-treated with flutamide. The present results demonstrate that cerebellar granule cells treated in vitro with testosterone are protected from oxidative stress via a mechanism mediated by the androgen receptor. Similarly to what we observed after in vivo administration of testosterone, the potentiation of the antioxidant defences seems to play a major role in the protection afforded by testosterone.