Mersalyl inhibited the respiration of heart mitochondria under conditions that required the transport of (-)-carnitine and acyl(-)-carnitines. The exchange of external carnitine and acylcarnitines for intramitochondrial carnitine was also inhibited by mersalyl and 1 mM mersalyl proved suitable for the inhibitor-stop assay of carnitine acylcarnitine translocase. The carnitine-carnitine and (-)-carnitine-acetyl(-)-carnitine exchanges involved a mole to mole exchange. The carnitine-carnitine exchange did not require energy. The carnitine acylcarnitine translocase resembles the Pi transport system in inhibition by mersalyl and N-ethylmaleimide and in lack of a cation requirement for activity; yet the two are not identical inasmuch as operation of only the former transport system was inhibited by long chain acyl(+)-carnitines. Additional results render it improbable that the transport of carnitine and acylcarnitines is catalyzed by any other known mitochondrial transport systems. The carnitine acylcarnitine translocase activity is unlikely to be shared by one of the carnitine acyltransferases because the mersalyl inhibition of carnitine palmitoyltransferase and carnitine acetyltransferase was noncompetitivcase. Rapid acetylation of intramitocondrial free (-)-carnitine occurred when acetyl-CoA was generated intramitochondrially but not with exogenous acetyl-CoA. Theese observations substantiate the view (Pande, S. V. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 883-887) that a system exists in mitochondria for the transport of carnitine and its esters and that the matrix has a pool of carnitine compounds which has access to that carnitine acyltransferase which is localized on the inner side of the inner mitochondrial membrane.