Cofactor requirements for the oxidation of palmitate-1-14C by 600 × g supernatant fraction of mouse skeletal muscle homogenate and by skeletal muscle mitochondria are described. Optimal oxidation of palmitate-1-14C by skeletal muscle mitochondria requires the presence of carnitine, ATP, CoA, and a Krebs cycle intermediate (e.g. succinate). Succinate, malate, alpha-ketoglutarate, and oxaloacetate are all equally effective in supporting the oxidation, but isocitrate is less effective. The oxidation of palmitate-1-14C by 600 × g supernatant fraction of muscle homogenate as well as by skeletal muscle mitochondria from dystrophic mice is significantly decreased compared with that of the normal littermate controls. The present results, together with the previous findings, suggest that the decrease in oxidation of palmitate-1-14C by the dystrophic muscle preparations is most likely due to a defect in one or more of the steps of the Krebs cycle.