Our preceding studies reported using animal liver microsomes that 2, 2', 3, 4, 4', 5'-hexachlorobiphenyl (hexaCB) (CB138), a worldwide and persistent organohalogen pollutant, was metabolized to two major hydroxy (OH)-metabolites, 3'-OH-CB138 (M-3) and 2'-OH-2, 3, 3', 4, 4', 5'-hexaCB (M-4), and two dechlorinated OH-metabolites (M-1 and M-2) in guinea pigs at much faster rate than in rats and hamsters. In this study, the distribution of four CB138 metabolites to the serum and liver 4 days after exposure and their fecal excretion were studied in guinea pigs administered with CB138 intraperitoneally. 3'-OH-CB138 (M-3) was a major metabolite in the liver, serum and feces. M-1 was observed as a minor metabolite in guinea pig feces. In contrast, trace amount of M-2 was present in guinea pig serum. However, 2'-OH-2, 3, 4, 3', 4', 5'-hexaCB (M-4) which was a major metabolite in the in vitro system using guinea pig liver microsomes was not found in all tissues and feces tested in this study. On the other hand, the exact chemical structures of M-1 and M-2 were determined to be 6'-OH-2, 3, 3', 4, 4'-pentaCB and 4'-OH-2, 2', 3, 4, 5'-pentaCB, respectively, by comparison of the retention time and mass fragmentation of the synthetic authentic samples in GC-MS. From these results, it is suggested that the metabolism of CB138 in guinea pigs may proceed by three pathways, a direct hydroxylation at 3'-position, and also the formation of 2',3'- or 3', 4'-epoxide and subsequent dechlorination and that three metabolites show the different mode of distribution and excretion.