ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca²⁺]_i and initiated downstream signalling cascades that involved ERK1/2, p38 and PI3-kinase. MAPK activation, the calcium response and induction of cell proliferation were inhibited by the ATP scavenger apyrase. 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 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 hypotonically-induced ATP release was blunted when the ATP-mediated signal transduction cascade was inhibited, i.e. purinergic receptors were blocked by suramin and PPADS, the Ca²⁺ response was inhibited by BAPTA, and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. 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.