The present work sets out to investigate how Ca²⁺ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca²⁺-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation of the release pathway. The accelerated inactivation is not observed in hypotonic Ca²⁺-free or high-K⁺ media. Addition of Ca²⁺-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca²⁺-activated K⁺ channels. The taurine release from control cells and cells exposed to Ca²⁺ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca²⁺-augmented taurine release. Exposure to Ca²⁺-mobilizing agonists prior to a hypotonic challenge also augments a subsequent swelling-induced taurine release even though the intracellular Ca²⁺-concentration has returned to the unstimulated level. The Ca²⁺-induced augmentation of the swelling-induced taurine release is abolished by inhibition of calmodulin, but unaffected by inhibition of calmodulin-dependent kinase II, myosin light chain kinase and calcineurin. The effect of Ca²⁺-mobilizing agonists is mimicked by protein kinase C (PKC) activation and abolished in the presence of the PKC inhibitor Gö6850 and following downregulation of phorbol ester-sensitive PKC isoforms. It is suggested that Ca²⁺ regulates the volume-sensitive taurine-release pathway through activation of calmodulin and PKC isoforms belonging to the novel subclass (nPKC).