A method is described for the estimation of in vivo activity of mitochondrial Acyl-CoA dehydrogenase, without the addition of extra ETF (electron transferring flavoprotein). 2. Using Fumarase as a marker enzyme, the activity of Acyl-CoA dehydrogenase was calculated per gram wet weight. 3. The enzyme displays negative cooperativity in fish muscle, but positive cooperativity in pig liver. S0.5 values for the substrate octanoyl-CoA are between 3 and 5 microM.