A large set of mutants of CYP102A1 from Bacillus megaterium have human cytochrome P450-like activities and the ability to metabolize a number of marketed drugs and steroids. Here, we tested whether the CYP102A1 mutants could be used to produce hydroxylated human metabolites of 17 beta-estradiol (E-2). A set of the mutants, which were generated by site-directed and random mutagenesis, was used to produce hydroxylated human metabolites of E-2 in this study. Some of the tested mutants could regioselectively generate 2-OH E-2 as a major metabolite but not other hydroxylated products. These results suggest that CYP102A1 mutants would be useful for the bioconversion of steroid hormones to hydroxylated products, which can be used for industrial applications.