We focusedon palmitoyl-CoA oxidized by two mitochondrial acyl-CoA dehydrogenases,very-long-chain acyl-CoA dehydrogenase (VLCAD) and acyl-CoA dehydrogenasefamily member 9 (ACAD9), both implicated in mitochondrial diseases.By combining MS and NMR, we identified the (2E)-hexadecenoyl-CoAas the expected α-β-dehydrogenation product and also the E and Z stereoisomers of 3-hexadecenoyl-CoA:a “γ-oxidation” product. The gene discussed is ACADVL; the disease is inborn mitochondrial metabolism disorder.