Objective— To determine whether S -nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Methods and Results— Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S -nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S -nitrosoglutathione reductase, which attenuated channel permeability. We measured S -nitrosoglutathione breakdown and NO _x concentrations at the myoendothelial junction and found S -nitrosoglutathione reductase activity to precede NO release. Conclusion— This study provides evidence for compartmentalized S -nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.