We tested the hypothesis that dietary alpha-linolenic acid (ALA) can exert effects on markers of cardiovascular risk similar to that produced by its longer chain counterparts in fish-oil. A dietary intervention study was undertaken to examine the effects of an ALA-enriched diet in 57 men expressing an atherogenic lipoprotein phenotype (ALP). Subjects were randomly assigned to one of three diets enriched either with flaxseed oil (FXO: high ALA, n = 21), sunflower oil (SO: high linoleic acid, n = 17), or SO with fish-oil (SOF n = 19) for 12 weeks, resulting in dietary intake ratios of n-6:n-3 PUFA of 0.5, 27.9 and 5.2, respectively. The relative abundance of ALA and EPA in erythrocyte membranes increased on the FXO diet (p < 0.001), whereas both EPA and DHA increased after fish-oil (p < 0.001). There were significant decreases in total plasma cholesterol within (FXO -12.3%, p = 0.001; SOF -7.6%, p = 0.014; SO -7.3%, p = 0.033) and between diets (p = 0.019), and decreases within diets after 12 weeks for HDL cholesterol on flaxseed oil (FXO -10%, p=0.009), plasma TG (-23%, p < 0.001) and small, dense LDL (-22% p = 0.003) in fish-oil. Membrane DHA levels were inversely associated with the changes in plasma TG ( p= 0.001) and small, dense LDL (p<0.05) after fish-oil. In conclusion, fish-oil produced predictable changes in plasma lipids and small, dense LDL (sdLDL) that were not reproduced by the ALA-enriched diet. Membrane DHA levels appeared to be an important determinant of these fish-oil-induced effects.