Although alkaline pH is known to trigger Ca(2+) influx in diverse cells, no pH-sensitive Ca(2+) channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca(2+)/Mg(2+) concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca(2+) concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La(3+)). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca(2+) levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca(2+) and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca(2+) permeable, suggesting that Ca(2+) influx through Cx43 HCs could account for the elevation in intracellular Ca(2+) elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca(2+) influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.