Singlet oxygen activates the mutagenicity of several benzo[a]pyrene (BP) derivatives in the absence of mammalian metabolic action. This has been demonstrated using a separated-surface-sensitizer system for generating chemically pure singlet oxygen, eliminating most of the complications that arise with singlet oxygen generation by conventional photosensitization. Salmonella typhimurium bacteria were exposed to singlet oxygen in the presence of certain BP derivatives and the mutation frequency determined with an azaguanine forward mutation assay. The mutation frequency was increased by exposure to singlet oxygen compared to light-only controls for those BP derivatives that were saturated at either the 7,8 or 9,10 positions but not both. The increase in mutation frequency depends on both the concentration of BP derivative and on the dose of singlet oxygen. Mutation frequency was also significantly increased when bacteria were treated with a solution of trans-7,8-dihydrodiol-BP that had been separately exposed to singlet oxygen, unequivocally demonstrating that the mutagenicity is due to the formation of a product of BP derivative oxidation by singlet oxygen and that this product has a lifetime at least on the order of minutes in acetonitrile. The requirement for singlet oxygen rather than some other form of reactive oxygen was confirmed by determination of the gas phase lifetime of the intermediate responsible for activating mutagenicity. This was performed by measuring the dependence of the mutation frequency on the distance separating the sensitizer from the target. This gives a value of 88 +/- 35 ms, which is in excellent agreement with the mean value of 89 ms calculated from previous independent determinations of the gas phase lifetime of singlet oxygen reported in the literature.