P2X receptors are membrane-bound ion channels that conduct Na?, K?, and Ca²? in response to ATP and its analogs. There are seven subunits identified so far (P2X₁-P2X?). P2X₂ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that P2X₂ receptor activation in PC12 cells couples to Ca²?-dependent release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at P2X₂ receptors. This leads to a hypothesis that P2X₂ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional P2X₂ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS (100 μM each), and exogenous exprerssion of ATP-binding mutant P2X₂ receptor subunit (P2X₂[K69A]). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride (10 μM), did not give any effects to neurite extension. The vesicular proton pump (H?-ATPase) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from P2X₂ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and P2X₂ receptors in hormoneinduced cell differentiation or neuronal synaptogenesis in local acidic environments.