We investigated regulation by extracellular ATP of channels important for volume regulation of rat hippocampal neurons. Cultures made from fetuses at the eighteenth gestational day were predominantly neuronal after 10–20 days in vitro, as indicated by immunostaining for neuron specific enolase. Neurons recorded with whole-cell patch clamp showed inward currents when membrane voltages were driven to values greater than −50 mV. Chloride conductance increased with 10 μM–100 μM extracellular ATP in a dose-dependent fashion. Similarly, an increase in taurine conductance was observed with 50 μM ATP. These currents were inhibited by the anion channel and purinergic receptor antagonists niflumic acid and suramin, respectively. The chloride conductance response to 10 μM ATP was increased over eight-fold in hypoosmotic medium (250 mOsm); however, chloride conductance in 0 mM ATP was not altered by this osmolality. Thus anion and osmolyte conducting channels activated via purinergic receptors may mediate volume regulation of hippocampal neurons.