We aimed to explore the effects of melatonin and n-3 polyunsaturated fatty acids (PUFA) supplementation on plasma and aortic nitric oxide (NO) levels in isoproterenol (Iso) affected spontaneously hypertensive (SHR) and Wistar rats. Untreated control rats were compared with Iso injected (118 mg/kg, s.c.) rats, and Iso injected plus supplemented with melatonin (10 mg/kg, p.o.) or PUFA (1.68 g/kg, p.o.) for two months. Plasma and aortic basal, L-NAME inhibited, adrenaline and acetylcholine stimulated NO were determined using Griess method. Plasma NO levels were lower in SHR versus Wistar rats. Iso decreased NO in Wistar while not in SHR. PUFA but not melatonin intake of Iso treated SHR increased plasma NO along with a decrease in systolic blood pressure. Basal aortic NO level was higher in SHR than Wistar rats and not altered by Iso. Intake of melatonin increased but PUFA decreased basal NO levels in Wistar+Iso and did not affect in SHR+Iso rats. Acetylcholine and adrenaline induced aortic NO release was significantly increased in Wistar+Iso but not SHR+Iso group. Melatonin intake increased Ach induced aortic NO in Wistar+Iso and SHR+Iso groups, whereas there was no effect of PUFA intake. Findings suggest that PUFA modulates plasma and melatonin aortic NO levels of isoproterenol affected rats in a strain-dependent manner.