Inwardly rectifying potassium (Kir) channels are a prominent feature of mature, postmitotic astrocytes. These channels are believed to set the resting membrane potential near the potassium equilibrium potential (EK) and are implicated in potassium buffering. A number of previous studies suggest that Kir channel expression is indicative of cell differentiation. We therefore set out to examine Kir channel expression in malignant glia, which are incapable of differentiation. We used two established and widely used glioma cell lines, D54MG (a WHO grade 4 glioma) and STTG-1 (a WHO grade 3 glioma), and compared them to immature and differentiated astrocytes. Both glioma cell lines were characterized by large outward K+ currents, depolarized resting membrane potentials (Vm) (−38.5 ± 4.2 mV, D54 and −28.1 ± 3.5 mV, STTG1), and relatively high input resistances (Rm) (260.6 ± 64.7 MΩ, D54 and 687.2 ± 160.3 MΩ, STTG1). These features were reminiscent of immature astrocytes, which also displayed large outward K+ currents, had a mean Vm of −51.1 ± 3.7 and a mean Rm value of 627.5 ± 164 MΩ. In contrast, mature astrocytes had a significantly more negative resting membrane potential (−75.2 ± 0.56 mV), and a mean Rm of 25.4 ± 7.4 MΩ. Barium (Ba2+) sensitive Kir currents were >20-fold larger in mature astrocytes (4.06 ± 1.1 nS/pF) than in glioma cells (0.169 ± 0.033 nS/pF D54, 0.244 ± 0.04 nS/pF STTG1), which had current densities closer to those of dividing, immature astrocytes (0.474 ± 0.12 nS/pF). Surprisingly, Western blot analysis shows expression of several Kir channel subunits in glioma cells (Kir2.3, 3.1, and 4.1). However, while in astrocytes these channels localize diffusely throughout the cell, in glioma cells they are found almost exclusively in either the cell nucleus (Kir2.3 and 4.1) or ER/Golgi (3.1). These data suggest that mislocalization of Kir channel proteins to intracellular compartments is responsible for a lack of appreciable Kir currents in glioma cells. © 2004 Wiley-Liss, Inc.