Studies were conducted to explore the mechanisms by which dietary fish oil decreases hepatic triglyceride secretion. Forty-five rats (15/group) were fed purified diets containing 10% fat as either fish oil, safflower oil or palm oil for 10 d. Plasma triglyceride concentration was lowest in the fish oil-fed group followed by the groups fed safflower oil and palm oil. The liver's capacity to oxidize fatty acids was assessed by assays of mitochondrial and peroxisomal beta-oxidation pathways in whole homogenates. Additionally, key enzymatic activities in the biosynthesis of triglyceride (diacylglycerol acyltransferase, phosphatidate hydrolysis) and phosphatidylcholine (CTP:phosphocholine cytidylyltransferase) were assayed. Compared with those fed palm oil the fish oil-fed animals showed 25% greater mitochondrial beta-oxidation but this difference was not statistically significant (P = 0.1). Fish oil feeding led to 45% greater (P less than 0.05) peroxisomal beta-oxidation. Diacylglycerol acyltransferase activity was unaffected by the type of dietary fat and slightly (13%) but significantly (P less than 0.02) lower cytidylyltransferase activity due to fish oil feeding was observed. More strikingly, both fish oil and safflower oil diets significantly lowered phosphatidate hydrolysis by 37 and 22%, respectively, compared with the palm oil diet. This activity directly correlated (r = 0.68; P less than 0.001) with plasma triglyceride concentration. Thus, dietary fish oil might suppress triglyceride secretion by decreasing glycerolipid synthesis, an effect mediated by changes in one or more enzymes involved in phosphatidate catabolism.