Aims: Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA: DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation. Methods and Results: EPA:DHA 6:1 and 9:1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA: DHA 3:1, 1:1, 1:3, 1:6, 1:9, EPA and DHA alone, and EPA:DHA 6:1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6:1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6:1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6:1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively. Conclusion: Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS.