The effects of erucic acid on the oxidative metabolism of rat-heart mitochondria have been investigated using intact animals, perfused beating heart, isolated mitochondria and mitochondrial extracts. Feeding rats with a diet containing erucic acid was found to lead to a diminished ability of the isolated heart mitochondria to oxidize various substrates, in accordance with previous reports (Houtsmuller et al., Biochim. Biophys. Acta 218 (1970) 564). This effect was almost pronounced with palmitylcarnitine as substrate, in which case the rate of oxidation was decreased by more than 50% at such a low erucic acid content in the diet as 1.4% given over 2-4 weeks. Oxidation of palmitylcarnitine was also found to be inhibited when erucylcarnitine was added to isolated heart mitochondria from control animals, in agreement with earlier observations (Christophersen and Bremer, FEBS Lett. 23 (1972) 230; Biochim. Biophys. Acta 280 (1972) 506). The inhibition was accompanied by a decrease in the rate and extent of reduction of mitochondrial flavoprotein. Experiments with perfused beating rat-heart likewise revealed an inhibition of flavoprotein reduction, as well as nicotinamide nucleotide reduction, when erucate was added to the perfusing medium of the beating heart respiring with oleate--but not with octanoate--as substrate. These data together with those earlier published in the literature indicate that erucic acid may interfere with the enzyme system involved in the mitochondrial oxidation of long-chain fatty acids, probably at the level of acyl-CoA dehydrogenase. Kinetic data supporting this conclusion, obtained with extracts of rat-heart mitochondria containing the acyl-CoA dehydrogenase and electron-transferring flavoprotein system, are presented. The possible implications of these results for the known effect of dietary erucic acid in causing an accumulation of fat in the heart are discussed.