Human cytochrome P450 1B1 (CYP1B1) is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17beta-estradiol (E2) to 4-hydroxy E2 by CYP1B1 has been postulated to be an important factor in mammary carcinogenesis. The inhibition of recombinant human CYP1B1 by 2,2',4,6'-tetramethoxystilbene (TMS) was investigated using either the Escherichia coli membranes of recombinant human CYP1B1 coexpressed with human NADPH-P450 reductase or using purified enzyme. 2,2',4,6'-TMS showed potent and selective inhibition of ethoxyresorufin O-deethylation (EROD) activity of CYP1B1 with IC(50) values of 2 nM. 2,2',4,6'-TMS exhibited 175-fold selectivity for CYP1B1 over CYP1A1 (IC(50), 350 nM) and 85-fold selectivity for CYP1B1 over CYP1A2 (IC(50), 170 nM). However, inhibition of human NADPH-P450 reductase activity by 2,2',4,6'-TMS was negligible. The modes of inhibition by 2,2',4,6'-TMS were noncompetitive for CYP1A1 and CYP1B1. Moreover, 2,2',4,6'-TMS significantly suppressed EROD activity and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 or CYP1B1 gene expression in human tumor cells such as HepG2 and MCF-10A. Taken together, our results indicate that 2,2',4,6'-TMS is a potently selective inhibitor of human CYP1B1 as well as a suppressor of CYP1B1 expression and may be a valuable tool for determining enzyme properties of human CYP1B1.