Rotenone, an organic pesticide and potent mitochondrial complex I inhibitor, causes Parkinson-like neurodegeneration in rodents and is implicated in human Parkinson's disease. In this rapid report, rotenone induced a dose-dependent decrease in succinyl-coenzyme A (CoA) and increase in β-hydroxybutyryl-CoA in multiple human cell lines (IC(50) < 100 nM). Rotenone also inhibited [U-(13)C(6)]-glucose-derived [(13)C]-acetyl-CoA and [(13)C]-succinyl-CoA biosynthesis in SH-SY5Y neuroblastoma cells. These changes are compatible with a compensatory metabolic rearrangement. Stable isotope dilution liquid chromatography-mass spectrometry and CoA thioester isotopomer analysis provided insight into mechanisms of rotenone toxicity, which will facilitate the development of new biomarkers of mitochondrial dysfunction.