Objective: We identified two additional patients with short-chain acyl-coenzyme A (CoA), further characterized the clinical and biochemical features of this defect, and compared it with other fatty acid oxidation defects. Design: We have measured the in vitro short-chain acyl-coenzyme A dehydrogenase (SCAD) activity in six affected persons with the electron-transfer flavoprotein-linked assay in the presence and absence of anti-medium-chain acyl-CoA dehydrogenase antibody. Urine organic acids, acylglycines, acylcarnitines, and radiolabeled substrate catabolism by skin fibroblasts were also examined. Results: All patients had some neurologic abnormalities, including hypotonia, hypertonia, or seizures. None of the patients had episodes of hypoglycemia; in the only patient tested, fasting ketogenesis was not impaired. Four patients were initially seen in the neonatal period, two with profound metabolic acidosis and two with mild acidemia; the other two cases were recognized in infancy. Enzymatic analysis of cultured skin fibroblasts demonstrated approximately 10% activity of SCAD when compared with control fibroblasts. Gas chromatography and mass spectrometry of urine revealed that ethylmalonic acid was present in all samples but not always at elevated concentrations; methylsuccinic acid and butyrylglycine were sporadically elevated. n-Butyrylcarnitine was often found in urine and plasma. Radiolabeled substrate metabolism was reduced to 40% to 60% of control values. Conclusions: Because affected persons do not consistently excrete characteristic metabolites, the diagnosis of this enzymatic deficiency is difficult. It is necessary to collect and analyze several urine and plasma specimens when the diagnosis is being considered in patients with neurologic abnormalities suggestive of this disorder. (J PEDIATR 1995;126:910-5)