The toxicities of polychlorinated biphenyls (PCBs) are thought to be mediated mainly by the aryl hydrocarbon receptor (AhR). However, little is known about changes to AhR-mediated effects caused by metabolic conversion of PCBs. To investigate whether hydroxylation affects the affinity of PCBs for the AhR, we measured the AhR agonistic activity of mono-hydroxylated PCBs (mono-OH-PCBs) and their non-hydroxylated analogs (PCBs) using yeast cells transduced with the human AhR and its response pathway. Fifty-two of 84 tested OH-PCBs and 12 of 24 PCBs exhibited AhR agonistic effects. Of 49 OH-PCBs that had the same chlorination patterns as the tested PCBs, 26 had activities that were more than twice those of their analogous PCBs, or became activated if their non-hydroxylated analogs were inactive. In particular, 3',4,5'-trichlorobiphenyl-2-ol and 3',4,4'-trichlorobiphenyl-3-ol were 37- and 22-fold more potent than their non-hydroxylated analogs and were 1.42 times and 1.08 times, respectively, as active as a standard, beta-naphthoflavone. The activities of only 5 OH-PCBs were reduced to less than half those of their non-hydroxylated counterparts. No tested PCBs were inactivated by the presence of a hydroxyl group. These findings underscore the need to rethink the toxicological evaluation of hydroxylated metabolites of PCBs and their abundance in the environment.