Adenine nucleotide translocase (AdNT) activity was studied in isolated mitochondria from normal rabbit aortas. The enzyme was inhibited by oleic acid, oleoylCoA, and oleoylcarnitine with 50% inhibition occurring at 5 muM, 6 muM and 14 muM, respectively (corresponding to 8, 10, and 23 nmol/mg protein). PalmitoylCoA and palmitoylcarnitine displayed similar potency to oleylCoA and oleoylcarnitine. The possibility that inhibition by fatty acid, acylCoA, and acylcarnitine could be attributed to non-specific detergency effects seems remote in that these compounds were more potent inhibitors of AdNT than equimolar concentrations of laurylsulfate. In addition, by use of the fluorescent probe N-phenyl-1-naphthylamine, it was shown that under the experimental conditions, inhibition of AdNT occurred at concentrations not exceeding a critical micelle concentration (CMC). Specificity was also suggested in that octanoylCoA was a weak inhibitor of AdNT and acetylcarnitine, butyrylcarnitine, cholesteryl oleate, and sphingomyelin were not inhibitory to the enzyme. In contrast to the observed inhibition of arterial AdNT by oleoylCoA and oleoylcarnitine, AdNT in isolated rabbit and rat heart mitochondria was inhibited only by oleoylCoA.