Simultaneous whole cell patch-clamp and indo 1 fluorescence measurements were used to characterize ATP-evoked membrane currents and intracellular Ca2+ concentration ([Ca2+]i) changes in isolated Hensen cells of the guinea pig organ of Corti. At negative holding potential, ATP activated a biphasic inward current and a concomitant increase in [Ca2+]i. The initial current activated within < 50 ms, showed a reversal potential near 0 mV and was reversibly inhibited by 30 microM suramin, suggesting this conductance was mediated by ATP-gated nonselective cation channels. The delayed ATP-activated current was mainly carried by Cl- as indicated by its shift in reversal potential when intracellular Cl- was replaced by gluconate. This Cl- conductance appeared to be Ca(2+)-activated secondarily to Ca2+ influx, since it required the presence of extracellular Ca2+ and was suppressed when an intracellular solution containing 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was used. In the absence of extracellular Ca2+, ATP still increased [Ca2+]i concomitant with a monophasic inward cation current, indicating Ca2+ release from intracellular stores. We conclude that Hensen cells have ionotropic and metabotropic P2 purinoceptors. They also have Ca(2+)-activated Cl- channels that can be activated by extracellular ATP, suggesting that purinoceptors in Hensen cells could play a regulatory role in ion and water balance of cochlear fluids.