Abstract These studies determine the levels of malate dehydrogenase isoenzymes in cardiac muscle by a steady state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of oxaloacetate. This inhibition is similar to that exhibited by lactate dehydrogenase in the presence of high concentrations of pyruvate. The results obtained by this method are comparable in resolution to those obtained by CM-Sephadex fractionation and by differential centrifugation for the analyses of mitochondrial malate dehydrogenase and cytoplasmic malate dehydrogenase in tissues. The use of standard curves of percent inhibition of malate dehydrogenase activity plotted against the ratio of mitochondrial MDH activity to the total of mMDH and cMDH activities [ m (m + c) malate dehydrogenase ratio] (percent m -type) is introduced for studies of comparative mitochondrial function in heart muscle of different species or in different tissues of the same species.