Physiological studies using P2X receptor agonists and antagonists have suggested that activation of P2X3 receptors by extracellular ATP depolarizes nociceptive neurons to evoke a sensation of pain (Bland-Ward and Humphrey, 2000). In order to trigger pain in response to ATP, purinergic receptors must reside in plasma membrane. Receptors insertion in plasma membrane is a dynamic process which is counterbalanced by receptor internalization by endocytotic mechanisms. Constant recycling of membrane- inserted fraction of P2X receptors has been suggested to be affected in neuropathic pain (Xu and Huang, 2004) and therefore represents an attractive target for pain treatment. In our work we studied trafficking of fluorescently tagged P2X3 receptors in cultured hippocampal neurons using Total Internal Reflection Fluorescence microscopy (TIRF). TIRF is an advanced optical microscopy method based on unique properties of evanescent field that is created on the surface of cell-bearing glass due to total internal reflection of the laser beam. Evanescent field allows selective visualization of those fluorescent molecules which are located in the immediate vicinity (~100 nm) of the plasma membrane of cultured cells (Pryazhnikov and Khiroug, 2007). In our study we selectively tracked perimembrane pool of P2X3 receptors which has greater probability of being inserted in the plasma membrane. We found agonist induced reduction of membrane associated pool of P2X3 receptors which is consistent with internalization process and may regulate the response of neurons to ATP. Bland-Ward PA, Humphrey PP. 2000. J Auton Nerv Syst 81:146–151. Pryazhnikov E., Khiroug L. 2008. Glia. 56(1):38–49. Xu GY, and Huang LY. 2004. Proc Natl Acad Sci U S A. 101(32):11868–73.