The biological consequences of cytoplasmic irradiation in mammalian cells are largely unknown. Using the microbeam at the Radiological Research Accelerator Facility, we examined cell lethality, mutation induction and the functional role of reactive oxygen species in human-hamster hybrid (AL) cells traversed by an exact number of alpha particles (150 keV/(m) through their cellular cytoplasm. Dual fluorochrome dyes and an image analysis system were used to determine the irradiation positions off the long ends of each cell. In comparison to nuclear irradiation (Hei et al., PNAS. 94: 3765,1997), cytoplasmic irradiation induced minimal toxicity such that traversal of cells with 4 alpha particles resulted in a surviving fraction of ~0.9. While cytoplasmic traversal with either 1 or 2 particles induced few S1- mutants in AL cells, those irradiated with 4 or more particles had an induced mutant fraction that was 2.5 fold higher than the background incidence. The particle delivery and imaging systems are so designed such that the chance of hitting the nucleus is only 0.4% with 4 (-particle traversals. In contrast to the multilocus deletions predominately induced by nuclear traversals, S1-mutants induced by cytoplasmic irradiation involved mostly small alterations similar to those of spontaneous origin. Concurrent treatment of cells with 8% DMSO significantly reduced the mutagenic potential of cytoplasmic irradiation. In contrast, pretreatment of cells with 10 mM of buthionine S-R sulfoximine, which reduces the cellular non-protein sulfhydryl levels to less than 5% of control levels, significantly enhances the mutagenic incidence by cytoplasmic irradiation. Our results show, for the first time, that cytoplasmic traversal of mammalian cells by (-particles is mutagenic via a mechanism different from that of nuclear traversal, probably involving oxyradicals.