The induction of DNA double-(dsb) and single-(ssb) strand breaks by RSU-1069, RSU-1131 and misonidazole in V79 mammalian cells has been investigated using sedimentation in isokinetic sucrose gradients after incubation for various times (1-3 hr) at 310 K under both hypoxic and aerobic conditions. Double strand breaks are produced by RSU-1069 and RSU-1131 predominantly under hypoxic conditions. Comparison of the cellular DNA damage induced by these agents leads to the following facts: (1) the yield of ssb induced by these agents is substantially increased under hypoxia, (2) RSU-1069 and RSU-1131 are much more effective than misonidazole, on a concentration basis, at causing strand breakage both under hypoxic and aerobic conditions; and (3) RSU-1069 is more efficient on a concentration basis than RSU-1131 at inducing both ssb and dsb under both conditions. From these findings and molecular studies it is suggested that these 2-nitroimidazole aziridines act as monofunctional alkylating agents under aerobic conditions, a factor that governs their aerobic cytotoxicity. Under hypoxic conditions, it is suggested that the induction of dsb and crosslinks by these agents (bifunctional character) may play a major role in determining the ability of such agents to act as hypoxia-selective cytotoxins.