ATP is released in many cell types upon mechanical strain, the physiological function of extracellular ATP is largely unknown, however. Here we report that ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca2+]i and initiated downstream signalling cascades that involved mitogen activated protein kinases (MAPKs) ERK1/2 and p38 as well as PI3-kinase. MAPK activation, the calcium response as well as induction of cell proliferation upon hypotonic stress were inhibited by preincubation with the ATP scavenger apyrase, indicating that hypotonic stress-induced signalling pathways are elicited by released ATP. Hypotonic stress increased prostaglandin E2 (PGE2) synthesis. Consequently, ATP release was inhibited by antagonists of PI3-kinase (LY294002 and wortmannin), phospholipase A2 (MAFP), cyclooxygenase-2 (COX-2) (indomethacin, etodolac, NS398) and 5,8,11,14-eicosatetraynoic acid (ETYA), which are involved in arachidonic acid metabolism. Furthermore, ATP release was abolished in the presence of the adenylate cyclase (AC) inhibitor MDL-12,330A, indicating regulation of ATP-release by cAMP. The hypotonic stress-induced ATP release was significantly blunted when the ATP-mediated signal transduction cascade was inhibited on different levels, i.e. purinergic receptors were blocked by suramin and PPADS, the Ca2+ response was inhibited upon chelation of intracellular Ca2+ by BAPTA, and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. In summary our data demonstrate that hypotonic stress initiates a feed forward cycle of ATP release and purinergic receptor signalling resulting in proliferation of prostate cancer cells.