Type I and type II spiral ganglion neurons (SGN) innervate the inner and outer hair cells of the cochlea, respectively. This neural system is established by reorganization of promiscuous innervation of the hair cells, immediately before hearing is established. The mechanism for this synaptic reorganization is unresolved but probably includes regulation of trophic support between the hair cells and the neurons. We provide evidence that P2X receptors (ATP-gated ion channels) contribute such a mechanism in the neonatal rat cochlea. Single-cell quantitative RT-PCR identified the differential expression of two P2X receptor subunits, splice variant P2X(2)(-3) and P2X(3), in a 1:2 transcript ratio. Downregulation of this P2X(2-3/3) receptor coincided with maturation of the SGN innervation of the hair cells. When the P2X(2-3) and P2X(3) subunits were co-expressed in Xenopus oocytes, the resultant P2X receptor properties corresponded to the SGN phenotype. This included enhanced sensitivity to ATP and extended agonist action. In P4 spiral ganglion explants, activation of the P2X receptor signaling pathway by ATPgammaS or alpha,betaMeATP inhibited BDNF-induced neurite outgrowth and branching. These findings indicate that P2X receptor signaling provides a mechanism for inhibiting neurotrophin support of SGN neurites when synaptic reorganization is occurring in the cochlea.