Our preceding studies have reported that 2,2',5,5'-tetrachlorobiphenyl (tetraCB)(CB52) is mainly metabolized to 3-hydroxy (OH)-metabolite by phenobarbital (PB)-inducible cytochrome P450 (P450) isoforms such as CYP2B1 and CYP2B18. In this study, the metabolism of CB52 by liver microsomes of untreated and PB-treated rabbits was investigated. Rabbit liver microsomes produced mainly 3-OH- and 4-OH-metabolites (M-1 and M-2) at an equal extent and two other metabolites (M-3 and M-4) and also that phenobarbital (PB) treatment accelerated the formation of all these metabolites, M-3 was assumed to OH-tetraCB by GC-MS. Another metabolite, M-4, was determined to 3,4-diOH-CB52 by GC-MS and 1H-NMR. Addition of antiserum against CYP2B4, a constitutive and PB-inducible rabbit P450 isoform, to a microsomal incubation system resulted in almost complete inhibition of the formation of 3-OH-, 4-OH- and 3,4-diOH-metabolites. These results suggest that CYP2B4 plays an important role in CB52 metabolism in rabbit liver.