beta-Oxidation of long-chain fatty acids increases many-fold in atherosclerotic aortas; this may be due to an increase in the activity of the mitochondrial enzyme hexadecanoyl-CoA: carnitine O-hexadecanoyltransferase EC 2.3.1.23 (trivial name: carnitine palmitoyltransferase, CPT). To investigate this possibility, an assay for arterial CPT was developed and used to measure CPT activity in mitochondrial fractions isolated from aortas of rabbits fed high-fat (HF) or high-fat plus cholesterol (HFC) supplemented diets. The arterial CPT assay was linear with respect to mitochondrial protein between 0.03 and 0.30 mg and assay time between 3 and 12 min. Maximum CPT activity was observed at concentrations of palmitoyl-CoA between 5 and 25 micron, higher concentrations of palmitoyl-CoA inhibited CPT activity. CPT activity was measured in mitochondrial fractions isolated from aortas of rabbits fed the HFC-supplemented diet for up to 48 days. No visible lesions were observed in aortas of rabbits fed HFC-diet for 3,9, or 21 days, however, by 48 days atheromatous lesions covered in excess of 60% of the intimal surface of the aorta. No lesions were visually observed in aortas of rabbits receiving the HF-diet. Despite the development of gross atherosclerotic lesions, there were no changes in CPT activity observed that could account for a dramatic increase in fatty acid oxidation. It is concluded that the increase in beta-oxidation of long-chain fatty acids in atherosclerosis is not attributable to an increase in CPT activity.