Deiters' cells function as supporting cells for the sensory-motor outer hair cells of the mammalian cochlea and are interconnected by gap junctions. Here the electrical and Ca2+ responses of Deiters' cells evoked by purinergic stimulation were investigated in the organ of Corti, the auditory sensory epithelium. Adenosine 59-triphosphate (ATP, 50-100 microM) applied focally by pressure increased the intracellular free Ca2+ concentration ([Ca2+]i). At the same time ATP evoked an early inward current that was followed by an outward component, reflecting a sustained Ca2+-dependent reduction of the pre-stimulus offset current. These responses were maintained when Ca2+ was removed from the extracellular medium (0 [Ca2+]o), indicating a contribution to Ca2+ signalling from P2Y metabotropic receptors. UV photolysis of caged inositol 1,4,5-triphosphate (InsP3, 16 microM) produced Ca2+ responses similar to those evoked by exogenous ATP, accompanied by reduction of the offset current. In Deiters' cells uncoupled by octanol (1mM), ATP activated only the early inward current, suggesting that functional gap junctions are required in the late phase of the current responses. Following the delivery of UV flashes to pairs of Deiters' cells loaded with caged InsP3, the electrical coupling ratio (CR), monitored by double patch-clamp recordings, was strongly attenuated. These data support the idea that, by promoting inflow of cations and by controlling gap-junction conductance in a Ca2+-and InsP3-dependent way, ATP might serve a protective role in the cochlea.