An examination has been made of some of the parameters which can affect mutant numbers in the Salmonella/microsome assay. The type of minimal media plates used for the assay and the concentration of glucose-6-phosphate, one of the co-factors necessary for mono-oxygenase action, had no effect on mutant numbers. Increases in mutated bacteria resulted from the use of (1) log-phase bacteria, (2) higher NADP concentrations than those normally recommended, and (3) higher phosphate buffer concentrations. Six mutagens, i.e., 2-acetylaminofluorene (AAF), 3,3'-dichlorobenzidine (3,3'-DCB), cyclophosphamide (CY), aflatoxin B1 (AFB1), 3-methylcholanthrene (3MC) and benzo[a]pyrene (BP), all requiring mono-oxygenase activation, were studied with two Salmonella typhimurium strains, TA98 and TA100, and liver preparations from rats given different inducing agent treatments using optimum conditions. Phenobarbitone induction was generally superior to Aroclor-1254 in converting these substrates to mutagens except for the polycyclic hydrocarbon substrates. A comparison of 3-methylcholanthrene, Aroclor-1254, beta-naphthoflavone or phenobarbitone as inducing agents revealed the first three of these to be equally effective in activating BP or 3MC to mutagens, whereas phenobarbitone was less active. Dual administration of 3-methylcholanthrene and phenobarbitone to rats did not result in an additive mutagenic effect using AAF, AFB1 or 3,3'-DCB as substrates, the numbers of mutant bacteria obtained being only equal to that seen with 3-methylcholanthrene alone. These differences were not due to there being different liver protein optima for the various inducing agent treatments. The foregoing results are discussed in relation to attempts to draw up a rigid protocol for mutagenicity testing.