The purpose of this study was to examine the effect of combining exercise with omega-3 fatty acids (n-3fa) supplementation on lipoprotein subfractions and associated enzymes. Subjects were 10 recreationally active males, aged 25 +/- 1.5 years (mean +/- SE), who supplemented n-3fa (60% eicosapentaenoic acid [EPA] and 40% docosahexaenoic [DHA]) at 4 g/d for 4 weeks. Before and after supplementation, subjects completed a 60-minute session of treadmill exercise at 60% Vo(2)max. Following a 24-hour diet and activity control period, blood was collected immediately before and after the exercise session to assess lipid variables: high-density lipoprotein cholesterol (HDL-C) and subfractions, low-density lipoprotein cholesterol (LDL-C) and subfractions and particle size, lecithin:cholesterol acyltransferase (LCAT) activity, and cholesterol ester transfer protein (CETP) activity. Supplementation with n-3fa alone increased total HDL-C and HDL(2)-C, while exercise alone increased total HDL-C, HDL(3)-C, and total LDL-C. LDL subfractions, particle size, and LCAT and CETP activities were not affected by supplementation. Combination treatment resulted in an additive effect for HDL(3)-C only and also increased LDL(1)-C versus baseline. LCAT and CETP activities were not affected by treatments. These results suggest that n-3fa supplementation or an exercise session each affect total HDL-C and subfractions but not LDL-C or subfractions. In addition, the combination of n-3fa and exercise may have additional effects on total HDL-C and LDL-C subfractions as compared to either treatment alone in active young men.