In the present study, we aimed to obtain further evidence in favour of the hypothesis that nitric oxide (NO) is a major mediator of endothelium-dependent vasorelaxation and to clarify whether NO plays a role in papaverine-induced vasorelaxation. The relaxant effects of acetylcholine (Ach), acidified NaNO2 or papaverine were investigated on isolated helical strips of the rat thoracic aorta precontracted with phenylephrine in an organ bath containing Krebs solution aerated with 95% O-2 and 5% CO2. The relaxation was quantified as % peak reduction of phenylephrine contracture. Saponin abolished the relaxant effects of Ach completely whereas it had no effect on the responses to acidified NaNO2 or papaverine. N-G-nitro-L-arginine (L-NOARG) reduced the effects of Ach significantly, but it was ineffective on the relaxation induced by acidified NaNO2. The inhibitory action of L-NOARG was partly restored by L-arginine, but not by D-arginine. Hemoglobin, hydroxocobalamin and hydroquinone exhibited significant inhibition on the relaxation evoked by Ach and acidified NaNO2. L-NOARG, hydroxocobalamin and hydroquinone caused only limited but significant decrease in the relaxation due to papaverine. This phenomenon was also observed by increasing phenylephrine concentration leading to an enhancement in the contraction. Our findings strongly support the view that Ach-induced relaxation of rat aorta strips is mediated by free NO released from the endothelium and the results suggest that NO may indirectly contribute to papaverine-induced relaxation.