Mutagenic activities of 1-, 2-, 3-, 4-, and 9-methylcarbazole were evaluated in S. typhimurium TA1535, TA1537, TA1538, TA98, and TA100. Only 9-methylcarbazole was found to be mutagenic in S. typhimurium TA100 in the presence of rat-liver homogenate. Mutagenic activity was also observed in TA100 for 2,9-, 3,9-, and 4,9-dimethylcarbazole. None of these methylated carbazole derivatives was mutagenic in TA1535, TA1537, TA1538 and TA98 in either the presence of absence of rat-liver homogenate. These results indicate that a 9-methyl substituent is associated with the mutagenic activity of these carbazole derivatives. Comparative studies on the mutagenic activity of 9-substituted carbazoles demonstrated that the activity of 9-ethylcarbazole was less than that of 9-methylcarbazole. 9-Phenyl- and 9-i-propylcarbazole were inactive under identical assay conditions. 9-Hydroxymethylcarbazole, a major metabolite of 9-methylcarbazole, was confirmed to be a direct-acting mutagen in S. typhimurium TA100. 9-Formylcarbazole was inactive as a mutagen when assayed with or without metabolic activation. These data are consistent with the finding that 9-hydroxymethylcarbazole is a major proximate mutagenic form of 9-methylcarbazole.