Previous work of our group has demonstrated that connexin (Cx) hemichannel (HC) responses can be activated by changes in intracellular calcium concentration ([Ca2+]i) in both Cx32 and Cx43 expressing cells. These [Ca2+]i-triggered responses are characterized by a bell-shaped response curve displaying activation of the responses with moderate [Ca2+]i transients (peak ~500 nM) and disappearance of the responses (further called inactivation) with [Ca2+]i transients peaking above ~500 nM. Here, we further explored [Ca2+]i-dependent inactivation of the HC responses. [Ca2+]i-triggered HC responses have been demonstrated to involve an intermediate step of calmodulin (CaM) and arachidonic acid (AA) signaling. Dye uptake assays with propidium iodide in C6-Cx43 cells revealed that AA-triggered responses were characterized by an S-shaped response curve with half-maximal activation occurring at ~330 mM, similar to the S-shaped curve observed for CaM activation with CALP1. AA-triggered dye uptake was inhibited by carbenoxolone and Gap 26, a Cx-mimetic peptide. The difference between S-shaped and bell-shaped response curves points to the absence of inactivation with the CaM or AA stimuli and suggests that the Ca2+-dependent inactivation is mediated by other mechanisms. We further performed experiments in which dye uptake through HC was stimulated with AA combined with simultaneous exposure to various concentrations of the Ca2+-ionophore A23187 to induce [Ca2+]i changes. A23187 concentrations in the range of 6 to 12 mM were used to incrementally induce Ca2+ transients peaking in the micromolar range. Dye uptake experiments revealed that AA-triggered HC responses were suppressed by a high [Ca2+]i in a dose-dependent manner, indicating that the bell-shaped [Ca2+]i-HC response curve is composed of an activation phase that involves CaM-AA signaling and an inactivation phase that is related to direct actions of Ca2+ on a yet unknown target.