The attenuated nitric oxide (NO) formation and/or elevated production of reactive oxygen species are often found in experimental and human hypertension. We aimed to determine possible effects of N-acetylcysteine (1.5 g/kg/day) and N-acetyl-5-methoxytryptamine (melatonin, 10 mg/kg/day) in adult spontaneously hypertensive rats (SHR) with established hypertension. After a six-week-treatment, blood pressure was measured and NO synthase (NOS) activity, concentration of conjugated dienes, protein expression of endothelial NOS, inducible NOS and nuclear factor-kappaB (NF-kappaB) in the left ventricle were determined. Both treatments improved the NO pathway by means of enhanced NOS activity and reduced reactive oxygen species level as indicated by decreased conjugated diene concentrations and lowered NF-kappaB expression. N-acetylcysteine (but not melatonin) also increased the endothelial NOS protein expression. However, only melatonin was able to reduce blood pressure significantly. Subsequent in vitro study revealed that both N-acetylcysteine and melatonin lowered the tone of phenylephrine-precontracted femoral artery via NO-dependent relaxation. Nevertheless, melatonin-induced relaxation also involved NO-independent component which was preserved even after the blockade of soluble guanylate cyclase by oxadiazolo[4,3-a]quinoxalin-1-one. In conclusion, both N-acetylcysteine and melatonin were able to improve the NO/reactive oxygen species balance in adult SHR, but blood pressure was significantly lowered by melatonin only. This implies that a partial restoration of NO/reactive oxygen species balance achieved by the antioxidants such as N-acetylcysteine has no therapeutic effect in adult rats with established hypertension. The observed antihypertensive effect of melatonin is thus mediated by additional mechanisms independent of NO pathway.