A previous study with aortic segments isolated from rats fed a fish oil-rich diet indicated an increase in acetylcholine-induced nitric oxide (^.NO)-mediated relaxation. However, it remained to be elucidated whether a fish oil-rich diet affected the vascular activity per se and the point of the ^.NO-cGMP pathway at which fish oil was acting. For this purpose two groups of Sprague-Dawley rats were fed a semipurified diet containing 5% lipids either corn oil (CO) or menhaden oil (MO) for 8 wk. We studied the mRNA and protein levels of endothelial ^.NO synthase (eNOS) and the NOS activity. The bioavailability of vascular ^.NO was assessed directly by electron spin resonance (ESR) spectroscopy. The levels of cGMP, L-arginine and L-citrulline were also evaluated in homogenates. The superoxide anion (O₂^.-) production and the related antioxidant activities were also studied in aortic segments. The aortic content of eNOS mRNA was increased in rats fed the MO-rich diet. This resulted in increases in both eNOS protein levels (70% in respect to the rats fed the CO-rich diet) and NOS activity (102%); the ^.NO production increased by 90%, cGMP levels by 100%, and L-arginine decreased by 30%. There was no increase in aortic O₂^.- production by dietary MO. The upregulation of the eNOS-cGMP pathway induced by dietary MO may contribute to the maintenance of vascular homeostasis and explain its beneficial effect in the prevention of arterial diseases.