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 liverin 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 μmmol/g liver) was 44% lower in the FO group relative to the SO animals. Thein vivo incorporation of [³H]glycerol into individual hepatic lipids resulted in triacyl‐glycerol/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 liverin vivo. This metabolic alteration was accompanied by a substantially lower amount (in μmol/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 [³H]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 providesin vivo evidence for a dampening effect of dietary fish oil on the synthesis of liver triacylglycerol relative to phospholipid and a moderate alteration ofde novo synthesis of individual phospholipids.