Gap junctions are specialized membrane structures that directly connect the cytoplasm of two neighbouring cells, allowing the passage of small molecules such as amino acids, ions and second messengers. Vertebrate gap junctions are composed of proteins, encoded by the connexin gene family and consist of two hemichannels, each one contributed by the cells that share this junctional channel. Several hemichannel opening stimuli are already known such as strong membrane depolarisation, low extracellular divalent cations, ischemia, metabolic inhibition and the production of reactive oxygen species. Recently, it was shown that elevation of intracellular calcium could trigger the opening of connexin 32 and connexin 43 hemichannels. Plotting intracellular calcium concentrations of connexin 43 expressing cells in function of the normalized ATP response shows a bell-shaped dose-response curve with a maximal response around 500 nM for HeLa-Cx43 cells and 750 nM for C6-Cx43 cells. This triggered ATP release is absent in C6-WT cells and in C6 cells stably transfected with pannexin 1. Incubating the cells with inhibitors of exocytosis, such as Botulinum toxin B, bafilomycin A1 and Tetanus toxin showed no inhibition of the calcium-induced ATP release. In this way, we could exclude vesicular mediated ATP release. The involvement of the P2X7 receptor pore could also be excluded via western blot analysis. All these data point to a strong involvement of connexin hemichannels. The molecular mechanism between increased [Ca²⁺]_i and opening of hemichannels is currently poorly understood. Our present work demonstrates that elevation of intracellular calcium activates calmodulin and Ca²⁺/CaMK-II. Furthermore, we could show that activation of calmodulin with CALP1, a calmodulin agonist, triggers ATP release in a dose-dependent way. This dose-reponse curve shows a sigmoidal function, indicating that calmodulin activation is responsible for hemichannel opening, while calcium is responsible for the closure of connexin hemichannels. Elevated intracellular calcium concentrations, together with activated Ca²⁺/CaMK-II activate and translocate cPLA2 from the cytoplasm to the nuclear envelope, leading to a release of aracidonic acid. This arachidonic acid is further metabolized by cyclooxygenases and lipoxygenases with the formation of reactive oxygen species. Blocking the arachidonic acid metabolic pathway with an inhibitor of cPLA2 (AACOCF3), a cyclogenase inhibitor (baicalein) or a lipooxygenase inhibitor (indomethacin) completely abolished the triggered ATP release, indicating that this pathway plays an important role in hemichannel opening. When we incubate the cells with free radical scavengers the opening of connexin hemichannels was inhibited. Elevating intracellular calcium can result in a conformational change of the CaM/NOS complex, leading to the production of NO. Incubating the cells with inhibitors of NO synthesis complete blocks the triggered ATP release. Our results indicated that calcium-triggered ATP release is mediated via the release of free radical species and NO. Further investigation will elucidate whether these free radical species directly influence hemichannels, or whether it is an indirect effect mediated by the oxidation of fatty acid, ultimately resulting in opening of connexin hemichannels.