Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl–currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl–currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200–500 μm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 μm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+(200–500 μm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl–. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.