The influence of dietary fish oil containing n-3 polyunsaturated fatty acids on the biosynthesis of triacylglycerol relative to total individual phospholipids was studied in rat liver in vivo. The dietary lipid (10% by weight of diet) was either sunflower oil enriched in linoleic acid (SO group) or MaxEPA fish oil/sunflower oil, 9:1 by weight (FO group) enriched in eicosapentaenoic acid (EPA, 20:5n-3) plus docosahexaenoic acid (DHA, 22:6n-3). After a 3-week feeding period, the triacylglycerol content (in mu-mol/g liver) was 44% lower in the FO group relative to the SO animals. The in vivo incorporation of [H-3]glycerol into individual hepatic lipids resulted in triacylglycerol/total phospholipid radioactivity ratios of 2.1 and 0.9 for the SO and FO groups, respectively. These results indicate an inhibitory effect of dietary EPA/DHA on triacylglycerol relative to phospholipid synthesis from intermediary 1,2-diacylglycerol in rat liver in vivo. This metabolic alteration was accompanied by a substantially lower amount (in mu-mol/g liver) of arachidonic acid and higher levels of EPA plus DHA in the triacylglycerol, choline glycerophospholipid (CGP), and ethanolamine glycerophospholipid (EGP) of the FO group. A moderately higher labelling of the EGP from [H-3]glycerol was observed in the FO as compared to the SO group (as evidenced by CGP/EGP radioactivity ratios of 1.3:1 and 1.8:1, respectively). The present study provides in vivo evidence for a dampening effect of dietary fish oil on the synthesis of liver triacylglycerol relative to phospholipid and a moderate alteration of de novo synthesis of individual phospholipids.