Anaplerosis from propionate was investigated in rat hearts perfused with 0-2mM [(13)C(3)]propionate and physiological concentrations of glucose, lactate, and pyruvate. The data show that when the concentration of [(13)C(3)]propionate was raised from 0 to 2mM, total anaplerosis increased from 5% to 16% of the turnover of citric acid cycle intermediates. Then, [(13)C(3)]propionate abolished anaplerosis from endogenous substrates, glucose, lactate, and pyruvate. Also, while the contents of propionyl-CoA and methylmalonyl-CoA increased with [(13)C(3)]propionate concentration, the content of succinyl-CoA decreased, presumably via activation of succinyl-CoA hydrolysis by a decrease in free CoA. Under our conditions, [(13)C(3)]propionate was a purely anaplerotic substrate since there was no labeling of mitochondrial acetyl-CoA, reflected by the labeling of the acetyl moiety of citrate.