To study the enzymatic basis of isovaleric acidemia, we have developed assay methods for isovaleryl-CoA and butyryl-CoA dehydrogenases that measure the amount of tritium released from the respective [2,3-3H]acyl CoAs. Because assay of these enzymes in human fibroblast homogenates was subject to interference by nonspecific reactions, we have isolated mitochondria from cultured skin fibroblasts by protease treatment, homogenization, and differential centrifugation. By using this assay method with these isolated mitochondria, we have demonstrated a specific deficiency of isovaleryl-CoA dehydrogenase [isovaleryl-CoA: (acceptor) oxidoreductase, EC 126.96.36.199] activity in cultured skin fibroblasts from five patients with isovaleric acidemia. In contrast, mitochondrial butyryl-CoA dehydrogenase [butyryl-CoA: (acceptor) oxidoreductase, EC 188.8.131.52] activity in these cells was preserved at normal levels. These results have been reproduced by using the conventional dye reduction assays. These observations give further support to the hypothesis that isovaleryl CoA is dehydrogenated by a specific enzyme and that isovaleric acidemia is due to a deficiency of this enzyme.