Cytochrome b5 (b5) is increasingly recognized to be of importance for specific cytochrome P450 (CYP) activities. We developed human b5/CYP-competent mutagenicity tester bacteria to study the role of b5 in the bioactivation activity of human CYP. These new tester bacteria were derived from the previously engineered human CYP-competent Escherichia coli K12 tester strain MTC, containing a bi-plasmid system for the co-expression of a specific CYP form (CYP1A2, 2A6 or 2E1) with human b 5, and human NADPH cytochrome P450 reductase (RED), resulting in the strain BTC-b5-1A2, BTC-b5-2A6 and BTC-b5-2E1, respectively. The relative content of b5 with CYP and RED in these three BTC-b5-CYP strains demonstrated physiologically relevant co-expression levels and typical CYP-specific activities could be determined with their specific chemical probes. These strains were applied in mutagenicity assays along with their corresponding b5-void strains to determine the effect of b5 on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivation of several promutagens. For CYP1A2, of the 5 compounds tested [2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3- methylimidazo(4,5-f)quinoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], only the mutagenicity of 2AA was slightly increased (∼1.5-fold) in the presence of b5. The CYP2E1- and CYP2A6-dependent mutagenicity of N-nitrosodiethylamine increased ∼3- and 23-fold, respectively when the bacteria contained b5. The CYP2A6-mediated mutagenicity of NNK increased ∼9-fold when co-expressed with b5. The stimulatory effect of b5 on the bioactivation of N-nitrosodi-n-propylamine was most striking. The mutagenicity of this procarcinogen was completely dependent on the co-expression of b5 with CYP2A6 or CYP2E1. This demonstrates the prominent role of b5 in the bioactivation of this carcinogen.