AIMS/METHODS: The calcium (Ca ²⁺)-switch technique was utilized to investigate the nature of the barrier governing (paracellular) permeability across the junctions of 'native' rabbit esophageal epithelium. This was done by mounting esophageal epithelium in Ussing chambers to monitor transepithelial electrical resistance (RT), a marker of junctional permeability. RESULTS: When exposed to Ca²⁺-free Ringer solutions containing EDTA, R_T declined ~35% below baseline over 2hrs, and this decline reversed within 2 hrs by restoration of (1.2mM) Ca²⁺- containing, normal Ringer solution ('Ca²⁺-switch technique'). Junctional resealing, i.e. increased R_T upon Ca²⁺ replacement, was assessed by the Ca²⁺-switch technique and shown to be: a) specific for Ca²⁺, with only Mn²⁺ among substituted divalent cations yielding partial resealing, b) a function of extracellular Ca²⁺ levels since maneuvers (BAPTA/AM or A23187 exposure) to alter intracellular Ca²⁺ had no effect; c) dose dependent, requiring as a minimum 0.5 mM Ca²⁺ and 1.2 mM Ca²⁺ for optimization and d) independent of protein synthesis since it was not inhibited by cycloheximide. Resealing was also inhibited by luminal antibodies or synthetic peptides to the extracellular domain of E-cadherin. Immunohistochemistry revealed E-cadherin within all layers of stratum corneum in Ca²⁺-free, but not Ca²⁺-containing, solution. CONCLUSION: The present investigation documents using the Ca²⁺-switch technique that esophageal epithelial junctions contain a major Ca²⁺-dependent component and that this component reflects adhesion between the extracellular domains of E-cadherin containing an HAV recognition sequence.