The effect of trans fat on the activities of liver mitochondrial and peroxisomal fatty acid oxidation enzymes was examined in various strains of rats. When Wistar and Sprague-Dawley rats were fed for 30 days diets containing either olive oil or partially hydrogenated corn oil as a source of cis- or trans-octadecenoate, respectively, the activities of various enzymes of mitochondrial and peroxisomal beta-oxidation measured with cis- and trans-9-octadecenoic acid as substrates showed little dietary fat-dependent change. In Fischer 344 rats, feeding trans fat for 15 mo increased only moderately various enzymes of beta-oxidation except for carnitine acyltransferase. The rate of mitochondrial ketogenesis and the activity of carnitine acyltransferase measured with trans-9-octadecenoic acid as a substrate were about half those with the cis-counterpart. Peroxisomes oxidized trans-9-octadecenoyl-CoA at a rate comparable to the cis-counterpart. It was concluded from this study and previous ones that the difference in the geometry of dietary fatty acid had only a marginal effect in modulating the hepatic fatty acid oxidation system, in spite of marked differences in the metabolic behavior of cis- and trans fatty acid in cell-free preparations and perfused liver.