Various biochemical properties of the adenine nucleotide translocase were compared with mitochondria prepared from control and host liver, and Morris hepatomas 7777, 7800 and 5123C. The transport of phosphoenolpyruvate on the adenine nucleotide translocase was found to be three to four times more active, and inhibition of the transporter by palmitoyl-CoA and atractylate considerably less in hepatoma the active transport of phosphoenolypyruvate was associated with a greater stimulation of calcium egress from the mitochondria matrix by the anion in the hepatoma. The diminished sensitivity of the adenine nucleotide translocase to palmitoyl-CoA in hepatoma mitochondria was associated with lower levels of long chain acyl-CoA esters in the whole tissue. A change in activation energy at 6 degrees C for the adenine nucleotide translocase was found in host liver mitochondria while no break point in the temperature curve was observed in hepatoma mitochondria. These results are most consistent with a change in the structure-function relationship of hepatoma mitochondria due to differences in lipid composition.