The effects of glutathione (GSH) and the combination of GSH and glutathione-S-transferase (GST) on aflatoxin B1 (AFB1) mutagenesis in the Salmonella mutagenicity assay using Salmonella typhimurium strains TA98 and TA100 were tested. Ten concentrations of AFB1 (0-1.0 micrograms/plate) were added to a liver microsomal homogenate (S9 mix) or to S9 mix containing GSH or S9 mix containing the combination of GSH + GST. One-third of the samples were plated directly. Two-thirds were incubated for 30 min at 37 degrees C prior to plating, and of those, half included bacteria. All samples were plated according to Ames et al . The results show that the addition of GSH and GSH + GST affected AFB1 mutagenesis by forming the AFB1-GSH conjugate and decreasing the availability of AFB1-8,9-epoxide. The effect of GST on GSH activity varied with the strain because of the different amounts of S9 mix used. The formation of the AFB1-GSH conjugate was verified by using reverse-phase high-performance liquid chromatography for quantitation of AFB1 and detection of AFB1-GSH.