The effect of supplementation with cod-liver oil containing eicosapentaenoic acid (EPA), 20∶5ω3, on bleeding times, thrombin-induced platelet aggregation, platelet protein, platelet cholesterol, and the level and fatty acid composition of individual phospholipids in the platelets of human subjects was determined. Measurement of these parameters was conducted before the subjects received the supplement (day 0), after they received the supplement for 14 days (day 14), and 14 days after the supplement was terminated (day 28) so as to monitor recovery. The mean bleeding times exhibited a marked increase (by 81%) with supplementation and returned to near basal (day 0) values within 14 days after the supplement was terminated. Cod-liver oil supplementation significantly reduced thrombin-induced platelet aggregation with a partial recovery being exhibited by day 28. The content of phospholipid, cholesterol and protein (μg/109 platelets) was not significantly different (P>0.05) when isolated from the subjects at day 0, 14 and 28, as neither were the composition of individual phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SPH)] given as % of total phospholipid. However, the fatty acid compositions of all platelet phospholipids were altered significantly by the fish oil supplement. In PC, EPA rose from 0.3 to 2.9% of total fatty acids and docosahexaenoate from 0.7 to 1.8% concomitant with a drop in arachidonate (from 14.1 to 9.6%) and linoleate (from 10.2 to 7.9%); these levels approached basal levels 14 days after supplementation was terminated. The highest percentage of EPA with supplementation was found in PE (4.3%), while the arachidonate fell from 38.8 to 30.5%, with low percentages of EPA occurring in PS (0.7%) and PI (0.5%). The level of 24∶1 in SPH increased significantly (from 17.8 to 24.8) with supplementation and reverted to basal values by day 28. These results suggest a close relationship of the observed fatty acid changes in individual platelet phospholipids to the altered hematological parameters and platelet-vessel wall interactions produced by cod-liver oil supplementation.