Thirty 18-month-old male F344/DuCrj rats were divided into the following groups: 10 untreated controls; eight vehicle-injected controls; and 12 ethanedimethanesulfonate (EDS)-injected rats. Untreated controls were killed immediately to check for testicular tumor incidence. In rats of the test group, a 75-mg/kg dose of EDS dissolved in dimethyl sulfoxide:water (1:3) was injected i.p. At intervals of 1, 2, 3, and 10 days after injection, two vehicle-injected control rats and three EDS-injected rats were sacrificed, and the testes were fixed by vascular perfusion. The midsagittal sections of all the fixed testes were examined to determine the incidence of macroscopic Leydig cell tumors, and some tumor tissues of the injection-treated groups were also investigated ultrastructurally. In 28 of 30 animals, a total of 78 Leydig cell tumors could be distinguished. Extensive and severe necrotic alterations accompanying fresh, multiple hemorrhages in early stages and reparative changes in later stages could be observed in a total of 78% of the 32 tumors examined from the EDS-injected group. The tumor cells exhibited ultrastructurally degenerative changes such as chromatin condensation and cytoplasmic vacuolation from 1 day after EDS injection. Therefore, EDS may be a necrotic agent for rat Leydig cell tumor.