It has been suggested that extracellular ATP signalling is implicated in protecting the auditory epithelium of the inner ear from noise-induced damage (Housley et al., 2009). Although ATP receptors have been found on several types of cells in the organ of Corti, these studies have focussed on either dissociated or a limited class of cells (Lagostena et al., 2001). Supporting cells show parallels with glia (Rio et al., 2002), however it is unclear if and how such cells, positioned near the first auditory synapse, might act in a protective manner. Multiple types of supporting cells are present and it is unknown whether ATP affects them similarly or activates different processes in them (Gale et al., 2004). To address these issues we have studied features of ATP-dependent signalling in the adult organ of Corti in an in situ system. The temporal bone of adult mice (ages P30-P90) was removed after killing the animals by cervical dislocation and the organ of Corti was exposed through a small opening at the apical end of the cochlea. For immunochemistry, antibodies against P2X2 (Alomone Labs) were used on 4% PFA fixed tissue. For calcium imaging, opened cochlear bones were incubated for 40 min in buffer containing 20 μM OGB1-AM, washed and then fluorescent signals were collected by widefield epifluorescence microscopy. Whole cell tight seal patch clamp recordings were made from inner sulcus and phalangeal cells identified visually. In some cases cells were decoupled with the gap junction blocker flufenamic acid (100 μM) to improve voltage clamp. There was extensive P2X2 immunoreactivity in the supporting cells of the organ of Corti. Comparatively, outer hair cells showed a limited degree of P2X2 on their apical surface. Consistent with these observations, 100 μM ATP puff applied to the organ of Corti produced a rise in intracellular (Cai) in supporting cells measured by OGB1. There was no detectable increase in Cai levels in the inner hair cells. ATP (100μM) applied to the inner sulcus and phalangeal cells surrounding the inner hair cells produced inward currents in both types of cells. The ATP gated currents showed inward rectification in both types of cells consistent with the properties of recombinant P2X channels. The ATP dependent currents in inner sulcus cells desensitized (τ1/2 = 1.6s) whereas little or no desensitization to prolonged application (15s) was observed in recorded phalangeal cells. We suggest that these results indicate differential expression of P2X receptor subunits and could reflect different roles for extracellular ATP signalling in these two cell types.