Methacrylonitrile (MAN) is an industrial chemical used to manufacture plastics and elastomers. The reported p.o. LD50 of MAN is 200 to 230 mg/kg in rats and 17 mg/kg in mice. Present investigations were undertaken to further characterize MAN metabolism and to study the metabolic basis for the species differences in its toxicity. Male F344 rats and B6C3F1 mice received a single gavage dose of 11.5 or 1.15 mg of [14C]MAN/kg and were placed in glass metabolism cages. Elimination of MAN in rats occurred primarily in expired air as unchanged MAN, acetone and CO2. Three major urinary metabolites of MAN were identified as N-acetyl-S-(2-cyanopropyl)-L-cysteine, N-acetyl-S-(2-hydroxypropyl)-L-cysteine and a deoxyuridine isomer. Quantitatively, rats and mice excreted ca. 7 and 49% of the high MAN dose in urine as N-acetyl-S-(2-hydroxypropyl)-L-cysteine, respectively. In addition, rats eliminated significantly more MAN-derived CO2 and deoxyuridine than mice. MAN elimination was almost complete within 24 hr after dosing and the tissue concentrations of MAN-derived radioactivity were, with the exception of the urinary bladder, consistently higher in rats than in mice. In conclusion, a quantitative difference between rats and mice in forming the epoxide intermediate, higher efficiency of mice to conjugate this intermediate with glutathione and greater capacity of rats to degrade it to acetone and CO2 are thought to contribute to the higher sensitivity of mice to MAN acute toxicity.