Medium chain acyl-CoA dehydrogenase (MCAD) and long chain acyl-CoA dehydrogenase (LCAD) deficiency are defects of mitochondrial beta-oxidation. The method of choice to measure specifically acyl-CoA dehydrogenase activity in human tissues uses purified electron transfer flavoprotein (ETF). We describe a simple and optimized method of purification allowing isolation of ETF with a degree of purity never reported so far. An assay for acyl-CoA dehydrogenase activity in cultured skin fibroblasts was developed using microquantities of electron transfer flavoprotein and substrate. MCAD deficiency was demonstrated in fibroblasts from nine patients and LCAD deficiency in fibroblasts from two patients.