Furan, which occurs in a wide variety of heat-treated foods, is a potent hepatotoxicant and liver carcinogen in rodents. In a 2-year bioassay, furan caused hepatocellular adenomas and carcinomas in mice and rats but also high incidences of bile duct tumors in rats. Furan is bioactivated by cytochrome P450 enzymes to cis-2-butene-1,4-dial, an α,β-unsaturated dialdehyde, which readily reacts with tissue nucleophiles. The objective of this study was to structurally characterize furan metabolites excreted with bile to better understand the potential role of reactive furan intermediates in the biliary toxicity of furan. Bile duct-cannulated F344/N rats (n = 3) were administered a single oral dose of 5 mg/kg b.wt. [(12)C(4)]furan or stable isotope-labeled [3,4-(13)C]furan, and bile samples collected at 30-min intervals for 4 h were analyzed by liquid chromatography-tandem mass spectrometry. A total of eight furan metabolites derived from reaction of cis-2-butene-1,4-dial with GSH and/or amino acids and subsequent enzymatic degradation were detected in bile. The main metabolite was a cyclic monoglutathione conjugate of cis-2-butene-1,4-dial, which was previously detected in urine of furan-treated rats. Furthermore, a N-acetylcysteine-N-acetyllysine conjugate, previously observed in rat urine, and a cysteinylglycine-glutathione conjugate were identified as major metabolites. These data suggest that degraded protein adducts are in vivo metabolites of furan, consistent with the hypothesis that cytotoxicity mediated through binding of cis-2-butene-1,4-dial to critical target proteins is likely to play a key role in furan toxicity and carcinogenicity.