The metabolism of dinitrobenzene (DNB) isomers in Fischer-344 rat, rhesus monkey and human erythrocytes was investigated. Erythrocytes from all species metabolized o-DNB and p-DNB to S-(nitrophenyl)glutathione conjugates although there were species differences in the rate and extent of conjugate formation. No metabolites of m-DNB were detected in the erythrocytes of any species. The rank order of the ability of the DNB isomers to produce methemoglobin in vitro varied from species to species, but p-DNB was always the most effective isomer. The data suggest that although the erythrocyte can conjugate DNB isomers with glutathione, this pathway offers no substantial protection from methemoglobinemia induced by dinitrobenzenes.