The contribution of membrane hyperpolarization to endothelium-dependent relaxations induced by acetylcholine was investigated in the femoral vein of the rat using a microelectrode technique and isometric tension recordings. Acetylcholine caused endothelium-dependent relaxations and hyperpolarization in tissues contracted with norepinephrine. The relaxation was sustained during a prolonged exposure to acetylcholine (less than or equal to 10 min). In contrast, the hyperpolarization declined with time. In the presence of nitro-L-arginine, a blocker of nitric oxide synthesis, the relaxation became smaller and transient, whereas the hyperpolarization was not affected. There was a temporal relationship between the relaxation and the hyperpolarization in the presence of nitro-L-arginine, when the two parameters were recorded simultaneously. In tissues contracted with 60 mM K+, in which hyperpolarization could not be observed, acetylcholine caused relaxations and these relaxations were abolished by nitro-L-arginine. The results suggest a contribution of both nitric oxide and membrane hyperpolarization to the endothelium-dependent relaxation induced by acetylcholine in the femoral vein of the rat.