Previous studies showed that binary mixtures of the environmental pollutants 1- and 3-nitrobenzo[a]pyrene produced a synergistic mutational response in the Salmonella reversion assay. Since nitroreduction is believed to mediate the direct-acting mutagenicity of the individual compounds, we have examined the role of nitroreduction in the mutagenicity of mixtures of 1- and 3-nitrobenzo[a]pyrene in the Salmonella plate incorporation assay. While mixtures of 1- and 3-nitrobenzo[a]pyrene induced up to 183% more revertants in strain TA98 than produced by equivalent amounts of the individual compounds, in the nitroreductase-deficient strain TA98NR the same mixtures only induced up to 57% more revertants than the individual compounds. Analysis of mixtures of 1- and 3-nitrosobenzo[a]pyrene (the two-electron reduction products of 1- and 3-nitrobenzo[a]pyrene) for mutation induction in TA98 yielded no evidence of a synergistic effect between the compounds. The mutagenicity of the mixtures was dependent upon the amount of the more mutagenic component. Salmonella cultures were also incubated with mixtures of 1- and 3-nitrobenzo[a]pyrene, as well as with equivalent amounts of the individual compounds. In two experiments, nitroreductive ability, as measured by the amount of 1-nitropyrene metabolized to 1-aminopyrene in 1 hr, was increased 9 to 105% in cultures pretreated with the mixtures as compared with cultures pretreated with the individual compounds. The results of this study support the hypothesis that nitroreduction is a major factor in the synergistic mutational response induced by 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium.