The activity of the adenine nucleotide translocator in rat heart mitochondria was quantitatively determined by the rate of [14C]ATP transport at 2 degrees C using the carboxyatractyloside inhibitor-stop technique. Linear uptake was obtained for 15 s, and with differing protein concentrations. The effect of matrix long-chain acyl-CoA esters upon the adenine nucleotide translocator activity was determined in these mitochondria. Incubation with palmitylcarnitine produced an increase in matrix long-chain acyl-CoA esters and decreased the velocity of [14C]ATP transport. Mitochondria isolated in the presence of KCN showed elevated levels of long-chain acyl-CoA esters, decreased transportable nucleotides, and very low adenine nucleotide translocator activity. Addition of potassium ferricyanide to these mitochondria caused a reduction in matrix acyl-CoA esters and partially restored adenine nucleotide translocator activity. Potassium ferricyanide also lowered matrix acyl-CoA in freshly isolated mitochondria and increased [14C]ATP transport. These findings show that the level of long-chain acyl-CoA esters within the mitochondrial matrix affects adenine nucleotide translocator activity and regulates mitochondrial activity.