Synthetic peptides homologous to the extracellular loops of the major vascular connexins represent a novel class of gap junction blockers that have been used to assess the role of direct cellular communication in arteries and veins. However, the specificity of action of such peptides on the coupling between smooth muscle cells (SMCs) has not yet been fully characterized. Isolated 3^rd order rat mesenteric arteries were therefore studied with respect to isometric tension (myography), [Ca²⁺]_i (Ca²⁺-sensitive dyes), membrane potential and input resistance (sharp intracellular glass electrodes). Confocal imaging was used for visualization of [Ca²⁺]_i events in individual SMCs in the arterial wall and membrane currents (patch-clamp) measured in individual SMCs isolated from the same arteries. A triple peptide combination (^37,43Gap 27 + ⁴⁰Gap 27 + ⁴³Gap 26) increased intercellular resistance (measured as input resistance) in intact arterial segments without affecting the membrane conductance of individual cells, and also interrupted electrical coupling between pairs of rat aortic A7r5 myocytes. In intact arterial segments the peptides desynchronized [Ca²⁺]_i transients in individual SMCs and abolished vasomotion without suppressing Ca²⁺ transients in individual cells. They also depolarized SMCs, increased [Ca²⁺]_i and attenuated acetylcholine-induced endothelium-dependent smooth muscle hyperpolarization. Experiments with endothelium-denuded arteries suggested that the depolarization produced by the peptides under basal conditions was in part secondary to electrical uncoupling of the endothelium from SMCs with loss of a tonic hyperpolarizing effect of the endothelium. Taken together, the results indicate that connexin-mimetic peptides block electrical signaling in rat mesenteric small arteries without exerting major non-junctional effects.