Inactivation of five distinct acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA (MCPA-CoA), the toxic metabolite of hypoglycin from unripe ackee fruit, was investigated using purified enzyme preparations. Short-chain acyl-CoA (SCADH), medium-chain acyl-CoA (MCADH) and isovaleryl-CoA (IVDH) dehydrogenases were severely and irreversibly inactivated by MCPA-CoA, while 2-methyl-branched chain acyl-CoA dehydrogenase (2-meBCADH) was only slowly and mildly inactivated. Long-chain acyl-CoA dehydrogenase (LCADH) was not significantly inactivated, even after prolonged incubation with MCPA-CoA. Inactivation of SCADH, MCADH and IVDH was effectively prevented by the addition of substrate. This mode of inactivation by MCPA-CoA explains the urinary metabolite profile in hypoglycin treated-rats, which includes large amounts of metabolites from fatty acids and leucine, and relatively small amounts of those from valine and isoleucine. Spectrophotometric titration of SCADH and MCADH with MCPA-CoA, together with the protective effects of substrate, indicates that MCPA-CoA is acted upon by, and exerts in turn irreversible inactivation of, SCADH and MCADH, confirming that MCPA-CoA is a suicide inhibitor (Wenz et al. (1981) J. Biol. Chem. 256, 9809-9812). Spectrophotometric titration data of LCADH and MCPA-CoA is typical of non-reacting CoA ester.