The effect of dichloroacetate (DCA) on the oxidation of medium-chain and short-chain fatty acids was studied in rat heart mitochondria. Dichloroacetate (1-10 mM) markedly decreased medium-chain and the short-chain fatty acid oxidation (oxygen uptake and release of 14CO2 from labelled fatty acids) by heart mitochondria; the oxidation of long-chain fatty acid, however, was unaffected by the compound. THe oxidation of acylcarnitines was not influenced by DCA. Dichloroacetate inhibited medium-chain and short-chain fatty acid oxidation by inhibiting the activity of mitochondrial acyl-CoA synthases. Inhibition of the enzyme activity by DCA was rapid and not due to its effect on pyruvate dehydrogenase.