1,3-Butadiene (BD) is used in the production of synthetic rubber and other resins. Carcinogenic effects have been observed in laboratory animals exposed to BD, with mice being more sensitive than rats. Metabolic oxidation of butadiene to epoxides is believed to be a crucial step in the initiation of tumors by BD. However, limited information is available that describes the in vivo metabolism of BD. Male Sprague-Dawley rats and B6C3F1 mice were exposed to 800 ppm [1,2 3,4-13C]butadiene for 5 h, and urine was collected during and for 20 h following exposure. Urinary metabolites were characterized using 1- and 2-dimensional methods of NMR spectroscopy. Three metabolites previously detected in vivo, N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine, N-acetyl-S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine, and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine, were present in both rat and mouse urine, accounting for 87% and 73% of the total metabolites excreted, respectively. A fourth metabolite, previously detected in vitro, 3-butene-1,2-diol, was also present in both rat and mouse urine and comprised 5% and 3% of the total metabolites excreted, respectively. An additional metabolite detected only in mouse urine that is derived from glutathione conjugation with epoxybutene was identified as S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine (4%). N-Acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine (4%), detected in mouse urine, is a thiohemiacetal product of 3-butenal. Additionally, mice excreted N-acetyl-S-(3-hydroxypropyl)-L-cysteine (5%) and N-acetyl-S-(2-carboxyethyl)-L-cysteine (5%), which could be derived from further metabolism of N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine or from glutathione conjugation with acrolein. Mice excreted N-acetyl-S-(1-(hydroxymethyl)-3,4-dihydroxypropyl)-L-cysteine (5%), which could be derived from glutathione conjugation with diepoxybutane (BDE), while rats excreted 1,3-dihydroxypropanone (5%), which may be derived from hydrolysis of BDE. These studies indicate that reactive aldehydes are produced as metabolites of BD in vivo, in addition to the reactive monoepoxide and diepoxide of BD. The greater toxicity of BD in mice compared with rats may be attributed to the greater ability of rats to detoxify BDE via hydrolysis, and/or to the production of reactive aldehydes.