The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y2 and P2Y4 receptor agonist, stimulated ATP release from Schwann cells in a dose-dependent manner with an ED50 of 0.24 µm. UTP-stimulated ATP release occurs through P2Y2 receptors as it was blocked by suramin which inhibits P2Y2 but not P2Y4 receptors. Furthermore, positive immunostaining of P2Y2 receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y4 receptors, did not significantly stimulate ATP release. UTP-stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP3 formation inhibitor lithium chloride, the cell membrane-permeable Ca2+ chelator BAPTA-AM and the endoplasmic reticulum Ca2+-dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi-complex inhibitor brefeldin A, microtubule disruption with nocodazole, F-actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP-stimulated ATP release is mediated by activation of P2Y2 receptors that initiate an IP3–Ca2+ cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.