It has been well known that 4-aminopyridine (4-AP) has an excitatory effect on vascular smooth muscle due to causing membrane depolarization by blocking K⁢-channel. However, we observed that 4-AP had an inhibitory effect on the mesenteric artery of rat. Therefore, we investigated the mechanism of 4-AP-induced vasorelaxation. The mesenteric arcuate artery and its branches were isolated and cut into ring. The ring segment was immersed in HEPES-buffered solution and its isometric tension was measured. 4-AP (0.1∼10 mM) induced a concentration-dependent relaxation, which was unaffected by NO synthase inhibitor, NG-nitro-L-arginine methylester (100μM) or soluble guanylate cyclase inhibitor, methylene blue (10μM). Glibenclamide (10μM), ATP-sensitive K⁢ channel blocker, did not exert any effect on the 4-AP-induced vasorelaxation. 4-AP relaxed the sustained contraction induced by 100 mM K⁢ or Ca2⁢ ionophore, A23187 (10μM) in a dose-dependent manner. In addition, 4-AP significantly decreased the phasic contractile response to norepinephrine in the absence of extracellular Ca2⁢. However, 4-AP did not block the 45Ca influx of rat aorta. From the above results, we suggest that 4-AP may not block the Ca2⁢ influx through Ca2⁢-channel, but act as a nonspecific vasorelaxant in arterial smooth muscle.