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The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorihated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp. strain LB400 (DHB[D.sub.LB400]), DHB[D.sub.P6]-I and DHB[D.sub.P6]-III from Rhodococcus globerulus P6, and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. strain RW1 (THB[D.sub.RW1]). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the [K.sup.app.m] values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorihated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3',5'-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophohic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHB[D.sub.P6]-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHB[D.sub.P6]-III than was DHB. Indeed, DHB[D.sub.P6]-III had the highest apparent specificity for 4,3',5'-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THB[D.sub.RW1] had the highest specificity for 2'-Cl DHB and was at least five times more resistant to inactivation by 2'-Cl DHB, consistent with the similarity between the latter and 2,2',3-trihydroxybiphenyl. Nonetheless, THB[D.sub.RW1] had the lowest specificity for 2',6'-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (k.sup.app.sub.cat] < 0.001 [s.sup.-1]).