An AHH-1 TK+/- cell derivative was developed that stably expresses human cytochrome P4501B1 (CYP1B1) cDNA in an extrachromosomal vector which confers resistance to 1-histidinol and co-expresses NADPH cytochrome P450 oxidoreductase (OR). The CYP1B1-expressing cell line was designated h1B1/OR. Microsomes prepared from CYP1B1 cDNA expressing cells exhibit elevated levels of 7-ethoxy-resorufin deethylase (EROD), 7-ethoxy-4-trifluoromethyl-coumarin deethylase (EFCD), benzo(alpha)pyrene hydroxylase (BPH), bufuralol 1'-hydroxylase, testosterone hydroxylase activities and spectrally quantifiable cytochrome P450. CYP1B1-containing microsomes did not contain detectable coumarin 7-hydroxylase, p-nitrophenol hydroxylase, lauric acid hydroxylase, (S)-mephenytoin 4'-hydroxylase or diclofenac 4'-hydroxylase activities. Kinetic parameters for selected substrates were compared among CYP1B1 and the two additional members of the CYP1 family, CYP1A1 and CYP1A2. For BPH and EFCD, the rank order of rates of substrate metabolism were CYP1A1 > CYP1B1 > CYP1A2. For EROD, the rank order of substrate metabolism was CYP1A1 > CYP1A2 > CYP1B1. For both EROD and EFCD the apparent K(m) values for CYP1B1 were more similar to CYP1A1 than to CYP1A2. In order to begin to characterize the promutagen activating ability of CYP1B1, the mutagenicity of selected chemicals was examined in h1B1/OR cells; there was increased sensitivity (CYP1B1-expressing relative to control cells) to the mutagenicity of benzo(a)pyrene, cyclopenta(c,d)pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and aflatoxin B1 (AFB). CYP1B1, expressed in this system, appears to be particularly efficient at activating AFB.