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Previous work demonstrated that human cytochrome P4501B1 (CYP1B1) forms predominantly 4-hydroxyestradiol (4-OHE2), a metabolite which is carcinogenic in animal models. Here, we present results from kinetic studies characterizing the formation of 4-OHE2 and 2-hydroxyestradiol (2-OHE2) by rat CYP1B1 using 17β-estradiol (E2) as a substrate. Km and Kcat values were estimated using the Michaelis–Menten equation. For rat CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 0.61 ± 0.23 and 1.84 ± 0.73 μM; the turnover numbers (Kcat) were 0.23 ± 0.02 and 0.46 ± 0.05 pmol/min/pmol P450; and the catalytic efficiencies (Kcat/Km) were 0.37 and 0.25, respectively. For human CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 1.22 ± 0.25 and 1.10 ± 0.26; the turnover numbers were 1.23 ± 0.06 and 0.33 ± 0.02; and the catalytic efficiencies were 1.0 and 0.30, respectively. The turnover number ratio of 4- to 2-hydroxylation was 3.7 for human CYP1B1 and 0.5 for rat CYP1B1. These results indicate that, although rat CYP1B1 is a low Km E2 hydroxylase, its product ratio, unlike the human enzyme, favors 2-hydroxylation. The Ki values of the inhibitor 2,4,3′,5′-tetramethoxystilbene (TMS) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.69 and 0.78 μM, respectively. The Ki values of 7,8-benzoflavone (α-NF) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.01 and 0.02 μM, respectively. The knowledge gained from this study will support the rational design of CYP1B1 inhibitors and clarify results of CYP1B1 related carcinogenesis studies performed in rats.