The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 × 10⁷ M), endothelium-dependent relaxations to A-23187 (10⁹-10⁶ M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10⁸-10⁵ M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10⁹-10⁵ M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME; 3 × 10⁴ M), and a cyclooxygenase inhibitor, indomethacin (10⁵ M), partially reduced A-23187-induced relaxation. However, in rings with valves, L-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3×10⁶ M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.