Liver and kidney organelles from rat and pig were separated by isopycnic sucrose density gradient centrifugation and located by marker enzymes. Carnitine palmitoyltransferase was shown to be exclusively a mitochondrial enzyme. In liver, approximately 52% of carnitine acetyltransferase activity was mitochondrial, 14% peroxisomal, and 34% located in a lipid-rich membranous fraction. Microsomes were a component of this last fraction and, when isolated by differential centrifugation, contained carnitine acetyltransferase activity. This enzyme has not previously been reported to be in peroxisomes. The specific activity of carnitine acetyltransferase in liver peroxisomes was two to three times greater than in the mitochondria or microsomes. Partial fractionation of broken rat liver peroxisomes into core, membranes, and the soluble matrix indicated that carnitine acetyltransferase had a similar distribution to the matrix enzyme, catalase. In gradients of rat and pig kidney, carnitine acetyltransferase was found primarily in the mitochondrial fractions. This enzyme was also not detected in microbodies, mitochondria, or microsomes from plants.

Carnitine acetyltransferase activity in liver fractions was confirmed by three separate assays—an 1(-)-carnitine-dependent release of coenzyme A (CoA) from acetyl-CoA, identification of the ¹⁴C-labeled reaction product acetylcarnitine, and the 1(-)-acetylcarnitine-dependent formation of acetyl-CoA from CoA. Carnitine acyltransferase activity for octanoyl-CoA in hepatic peroxisomes and microsomes was about equal to activity for acetyl-CoA. In the mitochondria, activity for octanoyl-CoA was six times greater than for acetyl-CoA.