The hepatocarcinogen and toxicant, furan, requires metabolic activation to elicit its toxic effects. The available experimental evidence indicate that the overall metabolism of furan is initiated via cytochrome P450 catalyzed oxidation to cis-2-butene-1,4-dial. This α,β-unsaturated dialdehyde reacts in vitro with protein and DNA nucleophiles. To determine if this compound is an in vivo intermediate in the metabolism of furan, rats were treated with either [ 12 C 4 ]furan or [ 13 C 4 ]furan and urine was collected for 24 h. Capillary LC/MS/MS analysis of the urine indicated that one of the metabolites was a mono-glutathione conjugate of cis-2-butene-1,4-dial. These results indicate that glutathione conjugation of the reactive metabolite of furan occurs in vivo. This metabolite may serve as a useful marker for furan exposure and metabolism in risk assessment studies.