The present experiments tested the hypothesis that decreased production of an endothelium‐derived relaxing factor in arteries of hypertensive animals contributes to impaired endothelium‐dependent relaxation. Acetylcholine‐induced relaxation of freshly isolated spontaneously hypertensive rat (SHR) vessels precontracted with norepinephrine was impaired compared with Wistar‐Kyoto rats (WKY). 10 μM indomethacin partially normalized the SHR response. Inhibition of nitric oxide synthesis with 100 μM. nitro‐L‐arginine methyl ester or 100 μM. NG‐monomethyl arginine shifted the acetylcho‐line relaxation curve to the right, but had no effect on the maximal relaxation in WKY and completely inhibited relaxation of SHR. A similar pattern was observed with methylene blue (0.3 μM). Acetylcholine‐induced relaxation of WKY vessels pre‐contracted with 5 μM norepinephrine and 100 mM K+ was attenuated 60% compared with vessels pre‐contracted with norepinephrine alone, and was completely inhibited by nitro‐L‐arginine methyl ester; relaxation of SHR vessels was decreased by 50%. Six‐hour storage at 37° C significantly attenuated acetylcholine‐induced relaxation in both strains; treatment with indomethacin improved the response. Moreover, relaxation of WKY vessels was completely inhibited by nitro‐L‐arginine methyl ester and NG‐monomethyl arginine after the storage period. The absence of L‐arginine‐induced relaxation and lack of effect of supplementation with L‐arginine indicated that the constitutively active NO synthetase was not induced and that L‐arginine substrate was limiting. The results indicate that mesenteric resistance arteries of the WKY express a relaxing factor generated by a non‐cyclo‐oxygenase, non‐nitric oxide synthetase pathway that is possibly a hyperpolarizing factor. This factor is either absent or expressed in very low amounts in vessels of the SHR. After a 6‐hour incubation period, expression of this factor is decreased and the nitric oxide pathway predominates. It is proposed that impaired relaxation of SHR resistance arteries may in part reflect the absence of this pathway.