In vitro incubations of purified rat cytochrome P-450 (CYP) 2B1 and human CYP 2B6 were performed to determine if CYP isozymes biotransform polychlorinated biphenyls (PCBs) enantioselectively. Enantioselective metabolism of chiral PCBs 45, 84, 91, 95, 132, and 136 and production of hydroxylated PCB metabolites (OH-PCBs) were observed, while no changes in PCB 183 atropisomer composition were observed for either isozyme. Enantiomer fractions (EFs) of parent PCBs, individually incubated as racemates at 25 ng/mL initial concentration, with rat CYP 2B1 ranged from 0.353 to 0.822. Enantioselectivity was also observed for PCBs 45 (EF = 0.437) and 132 (EF = 0.537) incubated at that concentration with human CYP 2B6. Both atropisomers of chiral PCBs appeared to be biotransformed simultaneously by rat CYP 2B1, except for (+)-PCB 132, but at different rates. Hydroxylated PCBs were identified using gas chromatography-high resolution mass spectrometry for all chiral PCBs enantioselectively transformed by CYPs. These metabolites did not correspond to any commercially available authentic standards, supporting the hypothesis that many unidentified OH-PCBs detected in wildlife may have arisen from in vivo biotransformation of chiral PCBs. A rough estimate suggested that more than half of the total congener metabolized by rat CYP 2B1 was converted to OH-PCBs. Similar concentration decreases were observed for congeners incubated with human CYP 2B6, but less OH-PCBs were formed. Formation of OH-PCBs via an enantioselective OH insertion mechanism was suggested, and may be a source of the unidentified OH-PCBs currently found in the environment.