Abstract 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 P2Y1 receptor with antisense oligonucleotide, as well as treatment with MRS2179 and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (P2Y1 antagonists), attenuates receptor-mediated PLC activity. Adenylyl cyclase inhibition by ADP remains unchanged under these conditions. Reverse transcription-PCR analysis showed that P2Y12 receptor is expressed in C6 cells. We therefore conclude that, in glioma C6 cells, two P2Y receptor subtypes are present: P2Y1, coupled to PLC, and P2Y12, negatively coupled to adenylyl cyclase.