The activities of hepatic fatty acid oxidation enzymes in rats fed perilla oil rich in alpha-linolenic acid (alpha-18:3) were compared with those fed saturated fats or safflower oil (the mixture of safflower oil and olive oil, 94:8, w/w) containing the same amount of polyunsaturated fatty acids with perilla oil exclusively as linoleic acid (18:2). When the rats were fed the diets containing 15% coconut, safflower, and perilla oils for 1 week, the rate of mitochondrial and peroxisomal oxidation of palmitoyl-CoA (16:0-CoA) in the liver homogenates was the highest in rats fed perilla oil. Among the rats fed the diets containing 15% palm, safflower, and perilla oils for 2 weeks, the rates of mitochondrial and peroxisomal oxidations of 16:0-, 18:2-, and alpha-18:3-CoAs were the highest in rats fed perilla oil, and the rate of oxidation of alpha-18:3-CoA by both pathways was higher than those of other acyl-CoAs in all groups. Dietary perilla oil relative to palm and safflower oils significantly increased the activities of carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase, and 2,4-dienoyl-CoA reductase. The substrate specificity of carnitine palmitoyltransferase appeared to be responsible for differential rates of the mitochondrial oxidation of acyl-CoAs. The substrate specificity of acyl-CoA oxidase did not account for the preferential peroxisomal oxidation of alpha-18:3 relative to 18:2. The preferential mitochondrial and peroxisomal beta-oxidation of alpha-18:3-CoA relative to 16:0- and 18:2-CoAs was also confirmed in rats fed laboratory chow irrespective of the substrate/albumin ratios in the assay mixture. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed a diet rich in alpha-18:3.