The effects of omega-3 fatty acid supplementation on high-density lipoprotein (HDL) subfraction distribution and composition were evaluated in five healthy volunteers taking 2.8 g/d of eicosapentaenoic acid (EPA) and 1.7 g/d of docosahexaenoic acid (DHA) for 6 weeks. This supplementation resulted in marked changes of the plasma fatty acid composition. Plasma total cholesterol (TC), HDL-cholesterol (HDL-C), and triglyceride (TG) levels did not change. HDL2-C increased by 74%, with a concomitant 19% decrease of HDL3-C; the HDL2 to HDL3 mass ratio increased from 0.30 +/- 0.19 to 0.47 +/- 0.28. The increase of HDL2 was confirmed by nondenaturing polyacrylamide gradient gel electrophoretic separation of HDL subclasses, otherwise showing no change in HDL particle size. After omega-3 supplementation, both HDL2 and HDL3 became cholesteryl ester (CE)- and TG-enriched and free cholesterol (FC)- and phospholipid (PL)-depleted. The reported findings provide a useful adjunct to the antithrombotic potential of omega-3 fatty acids.