The mutagenic activity of a group of diazo dyes based on benzidine and its congeners was compared following metabolic activation of the dyes through sequential reduction and oxidation. The dyes were reduced by incubating them with either a suspension of rat cecal flora or a hamster S9 mix supplemented with flavin mononucleotide. The products of dye reduction were then subjected to oxidative metabolism by either Aroclor-induced rat liver S9 or by hamster liver S9; the resultant mutagenic activity was assayed with Salmonella typhimurium TA1538. Fifteen of the 17 compounds tested were mutagenic, and the degree of mutagenicity was affected by the activity of both the reduction and oxidation systems used. Purified dyes required a reductive step to become mutagenic, but several of the crude dyes did not. All the positive compounds, however, were more mutagenic when the reduction step was included. The mutagenicity of the purified dyes was equal to or greater than that of an equimolar amount of benzidine or appropriate benzidine congener. For the crude dyes, there were no consistent quantitative relationships between the mutagenicity of the dye and that expected from the benzidine moiety.