To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ([Ca2⁢]i) mobilization and cell proliferation were investigated. ATP-induced [Ca2⁢]i increased in a dose-dependent manner (10⁣7 M∼10⁣3 M). ATP-induced [Ca2⁢]i increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced [Ca2⁢]i increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative P2Y receptor antagonist, dose-dependently weakened ATP-induced [Ca2⁢]i mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.