The 3-thia fatty acids, tetradecylthioacetic acid and 3,10-dithiadicarboxylic acid are mitochondrion and peroxisome proliferators. Administration of these promotes an increased transport of endogenous fatty acids to these organelles and a higher capacity of beta-oxidation. Administration of 3-thia fatty acids to rats resulted in a significant decrease of the hepatic level of docosahexaenoic acid (DHA) (17-24%) and especially eicosapentaenoic acid (EPA) (40-80%) accompanied by increased gene expression of mitochondrial 2,4-dienoyl-CoA reductase and enoyl-CoA isomerase. The mitochondrial oxidation of EPA was increased more than 4-fold after administration of 3-thia fatty acids. EPA-CoA was a good substrate for mitochondrial carnitine acyltransferase-I and treatment with 3-thia fatty acids increased the activity 1.7-fold. DHA was a poor substrate for both mitochondrial and peroxisomal beta-oxidation. DHA-CoA was a very poor substrate for mitochondrial carnitine acyltransferase-I and -II, and the activity did not increase after treatment. However, the peroxisomal DHA-CoA oxidase increased 10-fold after 3-thia fatty acid treatment, whereas the peroxisomal EPA-CoA oxidase increased only 5-fold. In isolated hepatocytes, 16% of total metabolized EPA was oxidized and 76% was incorporated into glycerolipids, whereas DHA was oxidized very little. We conclude that under conditions of increased mitochondrial and peroxisomal proliferation by 3-thia fatty acids, a relatively higher oxidation rate of polyunsaturated n-3 fatty acids might result in a decreased hepatic level of EPA and DHA. Under these conditions DHA seems to be oxidized by the peroxisomes, whereas EPA, which can be oxidized in both organelles, is mainly oxidized by mitochondria.