Abstract The alkylating agent busulfan (Myleran) adversely affects spermatogenesis in mammals. We treated male mice with single doses of busulfan in order to quantitate its cytotoxic action on spermatogonial cells for comparison with effects of other chemotherapeutic agents, to determine its long-term effects on fertility, and to assessits possible mutagenic action. Both stem cell and differentiating spermatogonia were killed and, at doses above 13 mg/kg, stem cell killing was more complete than that of differentiating spermatogonia. Azoospermia at 56 days after treatment, which is a result of stem cell killing, was achieved at doses of over 30 mg/kg; this dose is below the LD 50 for animal survival, which was over 40 mg/kg. Busulfan is the only antineoplastic agent studied thus far that produces such extensive damage to stem, as opposed differentiating, spermatogonia. The duration of sterility following busulfan treatment depended on the level of stem cell killing and varied according to quantitative predictions based on stem cell killing by other cytotoxic agents. The return of fertility after a sterile period did not occur unless testicular sperm count reached 15% of control levels. Dominat lethal mutations, measured for assessment of possible genetic damage, were not increased, suggesting that the stem cells surviving treatment did not propagate a significant number of chromosomal aberrations. Sperm head abnormalities remained significantly increased at 44 weeks after busulfan treatment, however, the genetic implications of this observations are not clear. Thus, we conclude that single doses of busulfan can permanently sterilize mice at nonlethal doses and cause long-term morphological damage to sperm produced by surviving stem spermatogonia.