The mechanism by which intracellular Ca²⁺ concentration regulates the permeability of gap junctions comprised of Connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca²⁺ in the presence of the Ca²⁺ ionophore ionomycin effected a sustained elevation in the intracellular [Ca²⁺] that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa Fluor594 (IC₅₀ of 360 nM Ca²⁺_i). The Ca²⁺-dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins Cx43, Cx44, and Cx49. The intracellular Ca²⁺-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action, but not by inhibitors of Ca²⁺ + calmodulin-dependent Protein Kinase II, or Protein Kinase C. In experiments using HeLa cells transfected with a Cx43 C-terminus truncation mutant (Cx43²⁵⁷), cell-to-cell coupling was similarly decreased by an elevation of the intracellular [Ca²⁺] (IC₅₀ of 310 nM Ca²⁺) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of intracellular [Ca²⁺] regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the C-terminus of Cx43.