Reversions induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were measured in the Salmonella/microsome quantitative plate assay using various concentrations of Salmonella typhimurium strain TA1538. The number of induced revertants increased with an increasing number of bacteria/plate, but the variation in reversion frequency was not as great as the variation in bacterial concentration. The effects of bacterial concentration on reversion fixation, phenotypic expression, selection of his+ revertants, and the interaction of the mutagen with bacterial DNA were examined. Suspension cultures of TA1538 were exposed to N-OH-AAF and various dilutions were prepared and assayed for revertants/10(8) bacteria. Reversion frequencies were very dependent on bacterial concentration between approximately 0.2 X 10(8) and 2 X 10(8) bacteria/plate. Revertants/10(8) TA1538 were reduced above about 2 X 10(8) bacteria/plate, indicating that culture conditions limited reversion fixation, expression and/or selection at these concentrations. The number of spontaneous revertants/plate determined both from bacteria exposed to dimethyl sulfoxide in suspension culture and in the Salmonella/microsome assay were not greatly affected by bacterial concentration. To study the effect of bacterial concentration on the interaction of the mutagen with bacterial DNA, various concentrations of TA1538 were exposed to the same dose of N-OH-AAF. Both revertants/10(8) TA1538 and DNA adducts varied inversely with bacterial concentration. The effect of bacterial concentration on both reversion fixation and/or expression and on mutagen binding to DNA may influence reversion frequencies in the Salmonella/microsome assay.