Carnitine-H 3 or palmitate-C 14 incubated with heart muscle preparations was incorporated into a compound that had chromatographic behavior in several systems identical to that of palmitylcarnitine chemically synthesized from palmityl chloride and carnitine. Palmitylcarnitine biosynthesis from palmitic acid and carnitine was dependent upon ATP and CoA in addition to substrates and an enzyme preparation. In contrast, palmityl carnitine formation from palmityl CoA and carnitine did not require ATP or CoA. The following reaction, catalyzed by palmitylcarnitine transferase, was shown to be reversible: Palmitylcarnitine+ CoA⇌ palmityl CoA+ carnitine. General chemical and metabolic properties of palmitylcarnitine were defined. Addition of palmitylcarnitine to heart muscle mitochondria increased respiration more than did addition of palmityl CoA, suggesting that palmitylcarnitine can more readily contribute its acyl group to the fatty acid oxidase system than can exogenous palmityl CoA. Carnitine increased degradation of pa1mityl-lC 14 CoA and steary1-lC 14 CoA to CO 2 and increased total oxygen uptake in the absence of ATP if acyl CoA were present. Carnitine did not appreciably enhance respiration or increase conversion of pa1mitate-lC 14 to CO 2 in the absence of ATP but did augment palmitate oxidation, as previously reported, when ATP and CoA were added to the system. Results are consonant with the hypothesis that the catalytic stimulation by carnitine of long-chain fatty acid oxidation is mediated via acylcarnitine formation, with subsequent transfer of the acyl group to CoA at the site of the fatty acid oxidase system.