: Using a charged particle microbeam whereby individual cell's cytoplasm could be irradiated with a single or an exact number of α particles, cell lethality, mutation induction and the functional role of reactive oxygen species in mediating the mutagenic response in the human—hamster hybrid (A,L,) cell assay system were assayed. The results showed that cytoplasm irradiation induced very little toxicity such that traversal through the cytoplasm with 16 alpha particles resulted in a cell survival fraction of —0.76. While cytoplasmic traversal with one particles induced relatively few CD59,-, mutant cells, those irradiated with 8 or more particles had an induced mutant frequency that was —3 fold higher than the background incidence. PCR analysis showed that CD59,-, mutation induced by cytoplasmic radiation involved almost exclusively small alterations similar to those of spontaneous origin. Treatment of cells with dimethyl sulfoxide(DMSO) suppressed the mutant frequency induced by cytoplasmic irradiation, whereas buthionine S—R—sulfoximine (BSO) treatment which reduces intracellular glutathione level,increased the mutation frequency. These novel findings showed that the mechanism of mutation by cytoplasmic irradiation was heavily dependent on oxyradicals. Moreover, using IF 7 monoclonal antibody to detect 8—OHdG induction, the data indicated that cytoplasmic irradiation generate oxidative DNA damage in the nuclei of the target cells.