The n-6/n-3 fatty acid (FA) ratio has increased in the Western-style diet to approximately 10-15:1 during the last century, which may have contributed to the rise in cardiovascular disease (CVD). Prior studies have evaluated the effects on CVD risk factors of manipulating the levels of n-6 and n-3 FA using food and supplements or investigated the metabolic fate of linoleic acid (LNA) and alpha-linolenic acid (ALA) by varying the n-6/n-3 ratios. However, no previous studies have investigated the potential interaction between diet ratios and supplementation with eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). We used a factorial design approach with adults (n = 24) in a controlled feeding trial to compare the accretion of EPA and DHA into red blood cell membranes (RBC) by adding a direct source (algal oil supplement) of EPA and DHA in a diet with a 10:1 versus 2:1 ratio of n-6/n-3 FA. Subjects were randomized into 8-week crossover diet sequences and each subject consumed three of four diets [10:1, 10:1 plus supplement (10:1 + S), 2:1 and 2:1 + S]. LNA and ALA intakes were 9.4 and 7.7%, and 1.0 and 3.0% during the low and high ALA diets, respectively. Compared to the Western-style 10:1 diet, the 2:1 diet increased EPA by 60% (P < 0.0001) in RBC membranes without the direct EPA source and a 34% increase (P = 0.027) was observed with the 10:1 + S diet; however, DHA levels increased in both diet ratios only with a direct DHA source. Shifting towards a 2:1 diet is a valid alternative to taking EPA-containing supplements.