We previously showed that, in the perfused rat heart, the capacity of n-fatty acids to generate mitochondrial acetyl-CoA decreases as their chain length increases. In the present study, we investigated whether the oxidation of a long-chain fatty acid, oleate, is inhibited by short-chain fatty acids, acetate or propionate (which do and do not generate mitochondrial acetyl-CoA, respectively). We perfused rat hearts with buffer containing 4 mM glucose, 0.2 mM pyruvate, 1 mM lactate, and various concentrations of either (i) [U-(13)C]acetate, (ii) [U-(13)C]acetate plus [1-(13)C]oleate, or (iii) unlabeled propionate plus [1-(13)C]oleate. Using mass isotopomer analysis, we determined the contributions of the labeled substrates to the acetyl moiety of citrate (a probe of mitochondrial acetyl-CoA) and to malonyl-CoA. We found that acetate, even at low concentration, markedly inhibits the oxidation of [1-(13)C]oleate in the heart, without change in malonyl-CoA concentration. We also found that propionate, at a concentration higher than 1 mM, decreases (i) the contribution of [1-(13)C]oleate to mitochondrial acetyl-CoA and (ii) malonyl-CoA concentration. The inhibition by acetate or propionate of acetyl-CoA production from oleate probably results from a competition for mitochondrial CoA between the CoA-utilizing enzymes.