In this study we characterized the subtypes of nucleotide P2Y receptors that respond to ADP in glioma C6 cells. Direct visualization of phosphatidylinositol 4,5-bisphosphate at the cell surface revealed that extracellular ADP activates phospholipase C (PLC). Knock-down of P2Y(1) receptor with antisense oligonucleotide, as well as treatment with MRS2179 and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (P2Y(1) antagonists), attenuates receptor-mediated PLC activity. Adenylyl cyclase inhibition by ADP remains unchanged under these conditions. Reverse transcription-PCR analysis showed that P2Y(12) receptor is expressed in C6 cells. We therefore conclude that, in glioma C6 cells, two P2Y receptor subtypes are present: P2Y(1), coupled to PLC, and P2Y(12), negatively coupled to adenylyl cyclase.