Ouabain, a specific inhibitor of the Na,K-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells (SMCs) is regulated through an interaction between the Na,K-pump and the Na,Ca-exchanger leading to an increase in the intracellular calcium concentration in discrete areas near the plasma membrane. The intracellular calcium concentration in individual SMCs was imaged in cultured rat aortic SMCs (A7r5) and simultaneously with isometric force in rat mesenteric small arteries. Paired A7r5 cells were used as a model for electrical coupling of SMCs by measuring membrane capacitance (Cm). SMCs were uncoupled (evaluated by inhibition of vasomotion and desynchronization of calcium transients in vascular wall, or by reduction to half of Cm measured in paired A7r5 cells) when the Na,K-pump was inhibited either by a low concentration of ouabain or by ATP depletion. Uncoupling with ouabain was associated with a localized increase of intracellular calcium in discrete sites near the plasma membrane. Reduction of Na,K-pump activity by removal of extracellular potassium also uncoupled cells, but only after inhibition of K-ATP channels. Inhibition of the Na,Ca-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular sodium and clamping low intracellular calcium concentration prevented the uncoupling. Immunohistochemical analysis suggested that a ouabain-sensitive α2 isoform of the Na,K-pump involved in this interaction. The experiments suggest that the Na,K-pump may affect gap junction conductivity via localized changes in intracellular calcium concentration through modulation of Na,Ca-exchanger activity.