1. Hensen's cells in the isolated cochlea were stimulated by extracellular adenosine 5'-triphosphate (ATP) applied to their endolymphatic surface while changes in membrane current and intracellular calcium concentration ([Ca2+]i) were measured simultaneously. The response consisted of (i) an initial rapid inward current accompanied by elevation of the [Ca2+]i, (ii) a more slowly rising inward current accompanied by a rise of the [Ca2+]i and (iii) a slowly developing reduction of input conductance. 2. The slower responses were maintained in the absence of extracellular Ca2+. Similar responses were produced by increasing the [Ca2+]i via UV flash photolysis of intracellular D-myo-inositol 1,4,5-trisphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester (caged InsP3) loaded at pipette concentrations of 8-16 microM. 3. The slow inward current, reversing around 0 mV, was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). 4. Bath application of U-73122 (1 microM), a phospholipase C inhibitor, eliminated the slow Ca2+-release component of the response to ATP. It is proposed that the effects of ATP are mediated by the co-activation of ionotropic P2X and metabotropic P2Y receptors. 5. Immunohistochemistry using light and electron microscopy revealed that inositol 1,4,5-trisphosphate (InsP3) receptors delineate a network within the cells. 6. The coupling ratio (CR) between cell pairs measured in dual patch-clamp recordings was 0.356 +/- 0.024. The coupling reversibly decreased to 51 % of the control within 2 min of applying 100 microM ATP. Flash photolysis of 32 microM intracellular caged InsP3 and 1 mM caged Ca2+ reduced CR to 42 and 62 % of the control, respectively. 7. We propose that endolymphatic ATP via P2X and P2Y receptors can control intercellular communication amongst Hensen's cells by reducing gap junction conductance in a Ca2+- and InsP3-dependent manner.