Methapyrilene (MP), a sedating antihistamine, is a potent rat hepatocarcinogen which has been thought to be non-genotoxic on the basis of the negative results in a small number of short-term mutagenicity tests. The present studies show that MP is a moderately active mutagen in the L5178Y/TK +/-----TK-/- mouse lymphoma assay (MLA) in the presence of aroclor-induced rat-liver S9, and that it induces predominantly small-colony thymidine kinase-deficient (TK-/-) mutants of demonstrated chromosomal origin. 10 of 12 small colony TK-/- mutants analyzed by banded karyotype (230-band level of resolution) show aberrations to chromosome 11b, the known location of the single functional TK gene in these cells. The observed aberrations from nine of the mutants included insertions, deletions and translocations while the tenth mutant had highly rearranged, multiple copies of chromosome 11 segments. By varying the concentrations of the S9 protein and cofactors it was shown that our standard S9 composition was close to optimum for activating MP to a mutagen. The activity and stability of various lots of S9 prepared in-house or purchased from a contract laboratory revealed significant differences. The ability of 2 lots of in-house S9 to activate a standard concentration of MP increased rapidly over the first 4 weeks of liquid nitrogen storage then declined slowly over the next 16 weeks. Three separate lots of purchased S9 were essentially inactive for the first 2 weeks of liquid nitrogen storage then increased in activity thereafter; these were the only occasions in which MP was not mutagenic in our hands. The mutagenic activity of pyrilamine (PYR), a structurally related antihistamine which is far less carcinogenic in rats, but easily detected in short-term tests as being genotoxic, was also investigated in the MLA. PYR was slightly less mutagenic than MP over a comparable range of concentrations, and also induced predominantly small-colony mutants. These studies fail to adequately explain the great carcinogenic differences between these two compounds, but are consistent with the potent hepatocarcinogenicity of MP resulting through a mutagenic mechanism.