N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN), which specifically induces bladder tumors, was mutagenic to Salmonella typhimurium strains TA1535 and TA100 in the presence of an S-9 mix prepared from the liver of rats treated with polychlorinated biphenyl. NADH was a more effective cofactor than NADPH in the activation of BBN by the rat liver S-9 fraction. N-Butyl-N-(3-carboxypropyl)nitrosamine, reported to be the main urinary metabolite of BBN as well as N,N-dibutylnitrosamine and to induce urinary bladder tumors specifically, was mutagenic without metabolic activation by the S-9 mix. The mutagenicities of 31 compounds related structurally or metabolically to BBN and N,N-dibutylnitrosamine were tested. Of these compounds, 13 were previously demonstrated to be carcinogenic, and 9 were noncarcinogenic. All carcinogenic compounds were mutagenic to strain TA1535 with or without the S-9 mix. Four of the 9 noncarcinogenic compounds were also mutagenic. These false positive compounds were predicted to be carcinogenic.