2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca(2+)]i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca(2+) influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca(2+)]i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca(2+)]i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca(2+)]i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca(2+) and SOC channel blockers such as SK&F96365, La(3+) and Gd(3+). PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca(2+)]i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca(2+)]i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.