Connexin 43 (Cx43) hemichannel (HC) responses can be activated by [Ca²⁺]_i changes according to a bell-shaped response curve with activation of the responses at moderate [Ca²⁺]_i transients (peak below ~500 nM) and disappearance of the responses (further called inactivation) with [Ca²⁺]_i transients peaking above ~500 nM. We further explored the mechanisms of [Ca²⁺]_i-dependent inactivation. [Ca²⁺]_i–triggered HC responses have been demonstrated to involve an intermediate step of calmodulin (CaM) and arachidonic acid (AA) signaling (see abstract & poster of De Vuyst E. et al.). ATP release assays on HC responses triggered in C6 glioma cells stably transfected with Cx43 cells by Ca²⁺-independent CaM activation with the peptide CALP 1 demonstrated an S-shaped concentration-response curve, indicating that high [Ca²⁺]_i related inactivation was absent with this trigger. Dye uptake assays (PI and EtBr) with AA-triggered responses revealed a similar S-shaped concentration-response behavior (half-maximal activation at ~330 mM). We next triggered HC dye uptake with AA and combined this trigger with various concentrations (6-12 mM) of the Ca²⁺-ionophore A23187 to induce large (mM) [Ca²⁺]_i transients. These experiments demonstrated concentration-dependent suppression of AA-triggered HC responses, confirming that high [Ca²⁺]_i levels inhibit HC responses. In a next step, we investigated whether the [Ca²⁺]_i- dependency of HC responses was related to Cx43 phosphorylation. Western blot analysis showed that A23187 concentrations activating HC responses reduced the degree of non-phosphorylated Cx43 protein (presumably due to the Ca²⁺-activated kinases) and that higher A23187 concentrations, that cause HC inactivation, restored the degree of non-phosphorylated Cx43. A possible explanation for this observation may reside in the activation at high [Ca²⁺]_i of a Ca²⁺-dependent serine/threonine protein phosphatase such as calcineurin. Inhibition of phosphatase type 2B with 2 mM cyclosporin A indeed reduced the high [Ca²⁺]_i-induced dephosphorylation and furthermore prevented inactivation of the HC responses probed with dye uptake. Our results demonstrate that the modulation of HC responses by [Ca²⁺]_i is a bi-modal process each governed by distinct signaling mechanisms.