The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca²⁺signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca²⁺ waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20–40 min. The peptides were shown to be more effective in inhibiting Ca²⁺ waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca²⁺ waves by connexin mimetic peptides did not affect the Ca²⁺ response to extracellular ATP. Although the intracellular Ca²⁺ response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca²⁺ signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca²⁺wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.