In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher concentration of cytosolic fraction. Studies on the glutathione-dependent reduction of BP mutagenicity in plate assays has shown that, in the presence of liver S9 concentrations greater than that required for optimal mutagenicity, the reduction in mutagenicity was related directly to the concentration of liver S9. Thus, in the Salmonella/microsome assay, when the concentration of rat-liver S9 was increased over and above the amount required for the optimal mutagenicity of BP, the mutagenic metabolites of BP were inactivated (by being trapped with cytosolic nucleophiles and/or by enzymic conjugation with glutathione); this effect increased more rapidly than their rate of formation. The concentration of liver S9 for optimal mutagenicity of test compounds requiring activation catalyzed by mono-oxygenases seems, therefore, to be related to the departure from linearity of the relationship between the rate of formation of mutagenic metabolites and the concentration of liver S9.