The mechanism by which intracellular Ca2+ concentration ([Ca2+]i) regulates the permeability of gap junctions composed 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 produced a sustained elevation in [Ca2+]i that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC50 of 360 nM Ca2+). 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 that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43Δ257), cell-to-cell coupling was similarly decreased by an elevation of [Ca2+]i (IC50 of 310 nM Ca2+) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca2+]i regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.