Connexin (Cx) hemichannels are closed before being incorporated into gap junctions but can be opened by various stimuli, thereby forming a release pathway for small paracrine messengers. Here, we investigated hemichannel-mediated ATP release in response to changes of intracellular Ca²⁺ ([Ca²⁺]_i) making use of C6 glioma cells stably transfected with Cx43 and primary glial cells isolated from rat cortex. The involvement of hemichannels was studied with gja1 gene silencing experiments and the exclusion of other release mechanisms. Hemichannel-related ATP responses appeared when [Ca²⁺]_i changes, brought about by exposure of the cells to the Ca²⁺ ionophore A23187, were in the 500 nM range while these responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and also glutamate activated a cascade sequentially involving calmodulin (CaM), calmodulin-dependent kinase II (CaMK-II), p38 mitogen activated kinase (MAPK), arachidonic acid (AA), reactive oxygen species (ROS) and NO. This cascade was also activated by stimulation at intermediate points such as activation of CaM with the Ca²⁺-like peptide (CALP1) or exogenous application of AA, and was also operational in primary glial cell cultures (astrocytes in majority) isolated from rat cortex. The disappearance of hemichannel responses with high [Ca²⁺]_i was independent of CaM signaling. We conclude that ATP release via Cx43 hemichannels can be invoked by [Ca²⁺]_i via an intermediate signaling axis that is combined with distinct OFF-mechanisms that help to limit and control the release process.