Extracts of human urine were shown to contain substances that strongly inhibited the liver S9-mediated mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Salmonella typhimurium TA98 strain in a liquid incubation assay. The inhibitory effect was unrelated to cytotoxicity and was similar with urine extracts from smokers and non-smokers. Under similar assay conditions, the mutagenicity of the related amino-imidazoazaarenes, 2-amino-3-methyl-imidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was also found to be strongly inhibited by urine extracts. Decreased or enhanced mutagenicity was seen with 2-acetyl-aminofluorene and 2-aminoanthracene depending on the type of assay, and the time of incubation in liquid medium. A weak inhibition of the mutagenicity of 2-nitrofluorene, a direct-acting mutagen, was observed only after a short incubation time. Mutagenicity of 4-nitroquinoline N-oxide was not altered by the presence of urine extracts at concentrations shown to be inhibitory for the mutagenicity of heterocyclic aromatic amines. Our data suggest that the inhibitory substances in urine act through their capacity to non covalently bind the parent heterocyclic and aromatic amines, thus affecting their availability in aqueous medium for diffusion into liver microsomes where metabolic activation takes place.