The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl^- and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins which may influence ClC-2 activity. Using immunoprecipitation of ClC-2 from HEK 293 cells stably expressing the channel, followed by electrophoretic separation of co-immunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin (GA) or radicicol, did not affect total amounts of ClC-2, but did reduce plasma membrane channel abundance. Functional experiments using the whole-cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the [Cl^-]_i dependent rightward shift of the fractional conductance. GA and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had opposite effects. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to [Cl^-]_i. This association may have important patho-physiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.