The O(6)-alkylguanine-DNA alkyltransferase inactivator O(6)-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea to study the role of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O(6)-alkylating agents. Exposure of the mice to 1, 3-bis(2-chloroethyl)-1-nitrosourea or O(6)-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O(6)-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O(6)-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O(6)-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1, 3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O(6)-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.