P2X4 receptors are found throughout the central nervous system, and studies have shown that these purinergic receptors are important regulators of alcohol intake. The ventral tegmental area (VTA) is an important region for the rewarding and reinforcing properties of alcohol, but the role of P2X4 receptors in this region is unknown. Using both immunohistochemical and electrophysiological methods, we examined the interaction between P2X4 receptors and alcohol on VTA neurons. Incubation of brain slices containing the VTA for 2 h with siRNA targeting P2X4 receptors resulted in about a 25% reduction in P2X4 immunoreactivity in tyrosine hydroxylase positive VTA neurons. In electrophysiological experiments, ATP (0.5-3 mM) produced a reduction in the spontaneous firing rate, and ethanol significantly reduced this inhibition. Exposure to siP2X4 for 2 h via the recording micropipette resulted in a suppression of the response of VTA neurons to ATP, but no significant reduction in the ethanol inhibition of the ATP response was observed after this P2X4 downregulation. These results support the idea that VTA neurons are inhibited by ATP, ethanol antagonizes this inhibition, and the ethanol-sensitive component of ATP inhibition is mediated by P2X4 receptors. This interaction of ethanol with P2X4 receptors may be an important regulator of the rewarding effects of ethanol, making P2X4 receptors an intriguing target for the development of agents to treat alcohol use disorders.