A sensitive assay for medium chain acyl-CoA dehydrogenase has been developed by substituting ferricenium hexafluorophosphate for the physiological acceptor, electron transferring flavoprotein. The ferricenium ion is a facile oxidant of the octanoyl-CoA-reduced enzyme with a Vmax of 1400 min-1 and a KM of 55 microM at pH 7.6. The ferricenium assay does not require additional mediator dyes, exhibits low background rates, and avoids the necessity of purifying substantial amounts of electron transferring flavoprotein. Unlike the fluorescence-based electron transferring flavoprotein assay, this new procedure can be performed aerobically. Both assays give comparable results when tested with crude fibroblast homogenates from normal and medium chain acyl-CoA dehydrogenase deficient patients. The convenience of the ferricenium method suggests it may be generally useful as a screening assay for a number of acyl-CoA dehydrogenases.