The ability of bovine bladder urothelial cells to activate genotoxic chemicals to mutagens was examined by cocultivating bladder cells with Chinese hamster V79 cells or Salmonella typhimurium as mutable targets. Activation of test chemicals to mutagenic intermediates by urothelial cells was detected by induction of 6-thioguanine resistance in V79 cells or by induction of histidine revertants in Salmonella. In the bladder cell-mediated V79 cell mutagenesis system, a significant increase in mutation frequency was induced by exposure to 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, and 4-aminobiphenyl were weakly mutagenic to V79 cells with bladder cell activation, while no mutagenic activity was detected with 1-naphthylamine, 2-naphthylamine, or benzidine. Because the mutagenic activity of the aromatic amines was low with V79 cells as the target, a bladder cell-mediated S. typhimurium system was developed for these chemicals. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, 4-aminobiphenyl and 2-naphthylamine were mutagenic to S. typhimurium TA98 and TA100 in the presence of bladder cells but not in their absence. Benzidine was mutagenic to TA98 but not to TA100. The putative noncarcinogen 1-naphthylamine was not mutagenic in the system. In contrast to the V79 data, 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine were not mutagenic with either bacterial strain. Mutagenic responses were related to both the number of bladder cells used for activation and the concentration of test chemical in the Salmonella assay. The data demonstrate that bovine bladder urothelial cells can activate carcinogens from three chemical classes to mutagens and indicate the different sensitivities of V79 cells and S. typhimurium to genotoxic agents.