ATP release into the extracellular milieu is an important growth stimulus for prostate cancer cells. In the present study hypotonicity-induced ATP release and the underlying signalling cascades were studied. Hypotonic incubation resulted in robust release of ATP, which was inhibited by cyclooxygenase-2- (COX-2) and phospholipase A2 (PLA2) inhibitors, the non- metabolizable arachidonic acid derivative ETYA as well as upon inhibition of adenylate cyclase (AC), thus suggesting an involvement of AC-cAMP-mediated signaling pathways. Hypotonic conditions resulted in upregulation of the chloride channels CLCN3, CLCN4, CLCN5 and CLCN6, which may be involved in the release of ATP, since ATP extrusion was abolished in the presence of the anion channel blockers niflumic acid, gadolinium, SITS and phloretin. Whole cell patch clamp measurements showed that hypotonicity increased anion channel conductance which was inhibited in the presence of DIDS, niflumic acid as well as the ATP-hydrolyzing enzyme apyrase. It is concluded that the ATP release upon hypotonic challenge is regulated by a cAMP-mediated signalling pathway with a contribution of ATP-permeable anion channels.