Most drug metabolizing cytochrome P450s (P450) are predominantly expressed in the liver. In contrast, humanCYP1B1is an extrahepatic P450 which is overexpressed in many tumours and has been strongly implicated in the activation of carcinogens. Rare allelic variants of theCYP1B1gene which encode an inactive protein have been identified. However, four polymorphisms which most likely do not abolish functionality have been described. In this report, we have characterized the functional consequences of these. A CYP1B1 cDNA, identical to a cDNA published previously, served as a template to introduce allelic changes either separately or in combination. The resulting effects on CYP1B1 activity were determined in membranes isolated fromEscherichia coliwhich coexpressed CYP1B1 together with P450 reductase. None of the allelic changes affected the CYP1B1 expression level. The allelic changes Arg48to Gly, Ala119to Ser and Asn453to Ser had little influence on theVmaxand theKmof the CYP1B1 mediated 2- and 4-hydroxylation of estradiol. In contrast, theKmof these metabolic pathways was increased at least three-fold by the allelic change Val432to Leu or by simultaneously changing Val432to Leu and Asn453to Ser. However, these alterations had little effect on the kinetic parameters of other CYP1B1 mediated reactions such as the epoxidation of (−)-trans-(7R,8R)-benzo[a]pyrene 7,8-dihydrodiol as determined by (r-7,t-8,t-9,c-10)-benzo[a]pyrene tetraol formation, or such as theO-dealkylation of ethoxyresorufin and the 1′-hydroxylation of bufuralol. Molecular modelling suggests that amino acid residue 432 of CYP1B1 may be involved in the interaction between CYP1B1 and P450 reductase. Since 4-hydroxyestradiol has been implicated in hormonal carcinogenesis and CYP1B1 is expressed in target tissues, the data presented demonstrate that polymorphisms in CYP1B1 have the potential to affect disease susceptibility.