To study the effect of the degree of unsaturation of dietary fatty acids on the production of free radicals and on the vascular smooth muscle tone in rings of the aorta, Sprague-Dawley rats were fed a semipurified diet containing 5% lipids from either corn oil (CO) or menhaden oil (MO) for 8 wk. The MO diet did not change the basal or NADPH-dependent superoxide anion (O·) release. There were no significant differences in phenylephrine-induced contractions between the two groups in intact rings. However, these contractions increased in endothelium-intact aortic rings from the MO group after addition of the nitric oxide (·NO) synthase inhibitor N^G-nitro-L-arginine and in endothelium-denuded rings, both indicating a greater endothelial basal ·NO production in rats fed with the MO diet. Endothelium-dependent relaxations in response to acetylcholine were more prominent in rings from the MO group. These differences were not due to an increased smooth muscle response to ·NO, because relaxations were the same using an exogenous ·NO donor. Our results indicate that dietary MO did not modify O· release by the vessel wall or relaxation due to the cyclooxygenase pathway, but it potentiated endothelial production of ·NO.