P2X receptors (P2XRs) are a distinct family of ligand-gated ion channels that are widely found throughout the peripheral and central nervous systems. They are fast acting, cation-permeable ligand gated ion channels that are gated by synaptically released extracellular ATP. P2X4Rs are the most abundant P2XR subtype expressed in the CNS and the most ethanol sensitive P2X subtype identified to date. Recent findings suggested that P2X4Rs may play a role in alcohol intake and preference in rats. Although, a growing body of evidence suggests a role for P2XRs in ethanol modulation, the sites responsible for the action of ethanol in these receptors are just beginning to be studied and remain unresolved. The present dissertation addresses this issue by using electrophysiology, molecular biology and molecular modeling strategies to identify ethanol sensitive molecular sites in these receptors. This dissertation sets three interrelated hypothesis build on each other: 1) Chapter 2 tests the hypothesis that the ectodomain-TM interface contains residues that are important for the action of ethanol in P2X4Rs; 2) Chapter 3 tests the hypothesis that Trp46 plays a role in ethanol and/or IVM modulation and 3) Chapter 4 tests the hypothesis that residues within the TM1 domain (residues 29 to 49) are important for the action of ethanol in P2X4Rs. The findings identified several ethanol sensitive amino acid residues and support the notion that there are multiple sites for ethanol action in P2X4Rs. In addition these studies increased our understanding of the sites and mechanisms of agonist transduction in P2X4Rs. Overall, the findings significantly add to our knowledge regarding the molecular sites for ethanol action in P2X4Rs and suggest possible targets for drug development to prevent or treat ethanol abuse and dependence.