DNA damage induced by various reactive oxygen species can be characterized using a set of repair endonucleases with defined substrate specificities. DNA damage profiles thus obtained in a cell-free system can be compared with those observed in cellular DNA. Using this approach, we have demonstrated that an illumination of Salmonella typhimurium cells with visible light in the presence of methylene blue gives rise to a DNA damage profile very similar to that of singlet oxygen in a cell-free system. Therefore, the genotoxicity observed under these conditions most probably is attributable to the direct action of this species. The damage consists mainly of base modifications that are subject to repair by uvrABC-independent pathways. Revertant frequencies observed in parallel in the strains TA100 and TA2638 indicate a pronounced mutagenicity of the lesions induced. Exposure of Salmonella typhimurium to tert-butylhydroperoxide gives rise to another form of damage profile that is also different from that produced by hydroxyl radicals in a cell-free system. However, the latter dissimilarity does not exclude hydroxyl radicals as ultimate reactive species, as a very rapid repair of the induced base modifications is observed, which might have distorted the damage profile despite immediate work up.