The hepatic metabolism of 2-chlorodibenzofuran was investigated in the rat. When 2-chloro[14C]dibenzofuran was intravenously administered to bile duct-cannulated rats, about 90% of radioactivity was excreted in bile and urine within 6 hr, the bile being the preferential route. Another group of rats received oral administration of cold 2-chlorodibenzofuran and bile fluid was collected by surgical bile duct cannulation for qualitative analysis. Three hydroxylated metabolites were isolated by high performance liquid chromatography from the bile fluid after hydrolytic digestion with sulfatase and beta-glucuronidase and were identified by mass and nuclear magnetic resonance spectrometries to be 2-chloro-3,7-dihydroxydibenzofuran, 2-chloro-3-hydroxydibenzofuran and 2-chloro-7-hydroxydibenzofuran, respectively. Analyses of the radioactive bile fluid by thin layer chromatography revealed that there was no detectable amounts of unmetabolized 2-chlorodibenzofuran in the bile fluid and the hydroxylated metabolites were present not as aglycons but as conjugated substances. The results suggest that 2-chlorodibenzofuran is rapidly metabolized in the rat, and the 3 and/or 7 positions play an important role in the metabolism of 2-chlorodibenzofuran.