Extracellular ATP has several neuro-humoral actions on cochlear physiology, many of which involve P2X receptor-mediated signal transduction. The present study extends the molecular physiology of P2X receptor gene expression in the cochlea to the principal platform for transgenic studies, the mouse model. P2X receptor subunits, which assemble to form ATP-gated ion channels, were localised in cryosections and whole-mount tissues from the adult mouse cochlea using a specific antiserum and immunoperoxidase histochemistry. Whole-cell voltage clamp recordings functionally correlated immunolocalisation of ATP-gated ion channels in isolated hair cells and supporting cells. P2X immunoreactivity was widespread throughout the epithelial lining of the cochlea (except vascular stria); spiral ganglion neurons, organ of Corti supporting cells, and outer hair cell (OHC) stereocilia exhibited strong P2X immunolabelling. Localisation of ATP-gated ion channels on the endolymphatic surface (cuticular plates and stereocilia) of outer hair cells was confirmed electrophysiologically. In contrast, Deiters' cells exhibited an even distribution of both immunolabelling over the whole cell membrane and inward currents could be evoked by localised ATP application anywhere on these cells. In both OHC and Deiters' cells, the slowly-desensitising inward currents were blocked by the P2X-selective antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), compatible with P2X subunits contributing to the ATP-gated ion channels. Our immunohistochemical and functional localisation of P2X receptors in the mouse cochlea extends previous studies to verify and characterise extracellular ATP signalling in the cochlea and extends support for P2X receptor-mediated regulation of endolymphatic ionic homeostasis, sound transduction, auditory neurotransmission and cochlear mechanics.