OBJECTIVE To evaluate the effects of four weeks of fish oil supplementation on apolipoprotein B100 production and lipoprotein metabolism in normolipidaemic males. METHODS Very low density lipoprotein (VLDL) apolipoprotein B100 (apoB100) kinetics in ten healthy, white males, aged 22-43 y (mean 32 y) were investigated using 13C-leucine technique and gas chromatography-mass spectrometry before and after fish oil supplementation. METHODS All subjects received 10 g (1.8 g EPA, 1.2 g DHA)/d of fish oil concentrate for four weeks. RESULTS Fish oil supplementation resulted in a decrease of total plasma VLDL (mean +/- s.d. 1.11 +/- 0.41 vs 0.87 +/- 0.28 mmol/l, P < 0.05) and triacylglycerol concentrations (0.74 +/- 0.27) vs 0.48 +/- 0.21 mmol/l, P < 0.01). VLDL apoB100 pool size was decreased without alteration of the fractional synthetic rate but a significant decrease of apoB100 production (2.23 +/- 0.90 vs 1.54 +/- 0.52 mg/dl/h, P < 0.02). Following fish oil supplementation plasma concentrations of glucose and insulin as well as lipoprotein and hepatic lipase activities were unchanged. Fasting plasma concentrations of non-esterified fatty acid (NEFA) were decreased (0.45 +/- 0.12 vs 0.33 +/- 0.10 mmol/l, P < 0.05). CONCLUSIONS Dietary supplementation with fish oil in healthy males results in decreased VLDL-triacylglycerol concentrations through a decrease in VLDL particle synthesis. The decrease in NEFA substrate supply also contributes.