We have demonstrated that downregulation of proliferation by CD4(+) T-cells in mice fed n-3 PUFA diets is dependent on the involvement of CD28. Therefore, we hypothesized that the balance of co-stimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. Mice were fed a control corn oil (CO)-enriched diet devoid of n-3 PUFA, or diets enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 14d. The proliferation of splenic CD4(+) T-cells was suppressed by DHA and EPA following stimulation with anti-CD3 and anti-CD28. Surprisingly, the number of surface CD28 molecules was not reduced in activated CD4(+) T-cells from either group of n-3 PUFA-fed mice. However, in mice fed EPA, CTLA-4 protein levels were enhanced significantly 72 h post-activation (P<0.01). Therefore, we conclude that dietary EPA may suppress CD4(+) T-cell activation by enhancing the downregulatory co-receptor CTLA-4, while not altering the levels of CD28.