Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl- channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (VPost-RVD < Vbaseline). In glioma cells, RVD was largely inhibited by treatment with a combination of CP channel inhibitors, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and Cd2+ (VPost-RVD > 1.4*Vbaseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K +-Cl- cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd2+ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of CP channels vs. transporters to this process is ∼60-70% and ∼30-40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl- channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCCl and KCC3a. Copyright © 2005 the American Physiological Society.