At the vertebrate neuromuscular junction (nmj), ATP is known to be coreleased with acetylcholine from the synaptic vesicles. We have previously shown that the P2Y1 receptor is localized at the nmj. Here, we extend the findings to show that another nucleotide receptor, P2Y2, is also localized there and with P2Y1 jointly mediates trophic responses to ATP. The P2Y2 receptor mRNA in rat muscle increased during development and peaked in adulthood. The P2Y2 receptor protein was shown to become restricted to the nmjs during embryonic development, in chick and in rat. In both rat and chick myotubes, P2Y1 and P2Y2 are expressed, increasing with differentiation, but P2Y4 is absent. The P2Y2 agonist UTP stimulated there inositol trisphosphate production and phosphorylation of extracellular signal-regulated kinases, in a dose-dependent manner. These UTP-induced responses were insensitive to the P2Y1-specific antagonist MRS 2179 (2'-deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt). In differentiated myotubes, P2Y2 activation induced expression of acetylcholinesterase (AChE) protein (but not control alpha-tubulin). This was shown to arise from AChE promoter activation, mediated by activation of the transcription factor Elk-1. Two Elk-1-responsive elements, located in intron-1 of the AChE promoter, were found by mutation to act in this gene activation initiated at the P2Y2 receptor and also in that initiated at the P2Y1 receptor. Furthermore, the promoters of different acetylcholine receptor subunits were also stimulated by application of UTP to myotubes. These results indicate that ATP regulates postsynaptic gene expressions via a common pathway triggered by the activation of P2Y1 and P2Y2 receptors at the nmjs.