Cytochrome P450 (P450)-catalyzed oxidation of the aromatic ring of estradiol can result in 2- or 4-hydroxylation. Which of these products is formed is biologically important, as the 4-hydroxylated metabolite is carcinogenic, whereas the 2-hydroxylated metabolite is not. Most human P450 enzymes, including CYP1A1 and CYP1A2, exhibit a high preference for estradiol 2-hydroxylation, but human CYP1B1 greatly favors 4-hydroxylation. Here we show that heterologous expression of the human, monkey, dog, rat, and mouse CYP1B1 enzymes yields active proteins that differ in their estradiol hydroxylation specificity. The monkey and dog orthologs, like the human enzyme, preferentially catalyze 4-hydroxylation, but the rat and mouse enzymes favor 2-hydroxylation. Analysis of the CYP1B1 sequences in light of these findings suggested that one residue, Val395 in human CYP1B1, could account for the differential hydroxylation specificities. In fact, mutation of this valine in human CYP1B1 to the leucine present in the rat enzyme produces a human enzyme that has the 2-hydroxylation specificity of the rat enzyme. The converse is true when the leucine in the rat enzyme is mutated to the human valine. The role of CYP1B1 in estradiol carcinogenicity thus depends on the identity of this single amino acid residue.