The objective of this study was to determine the relative contribution of chloride channels to volume regulation of cultured rat cortical astrocytes following hypotonic cell swelling. Using a Coulter counter, we show that cortical astrocytes regulate cell volume by ~60% within 45 min following hypotonic challenge. This volume regulation was supported when chloride was replaced with bromide, nitrate, methanesulfonate or acetate, but was inhibited when chloride was replaced with isethionate or gluconate. Additionally, substitution of chloride with iodide completely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by chloride channel blockers, including NPPB, DIDS or niflumic acid. Surprisingly, combination of cadmium with NPPB, DIDS, or niflumic acid inhibited regulation to a greater extent than either of these drugs alone. Volume regulation did not differ among astrocytes cultured from different brain regions, as cerebellar and hippocampal astrocytes exhibited identical behavior to cortical astrocytes. This data suggests that chloride flux through ion channels rather than transporters is essential for volume regulation of cultured astrocytes in response to hypotonic challenge.