The inwardly rectifying potassium channel Kir4.1 is widely expressed by astrocytes throughout the brain. Kir4.1 channels are absent in immature, proliferating glial cells. The progressive expression of Kir4.1 correlates with astrocyte differentiation and is characterized by the establishment of a negative membrane potential (> −70 mV) and an exit from the cell cycle. Despite some correlative evidence, a mechanistic interdependence between Kir4.1 expression, membrane hyperpolarization, and control of cell proliferation has not been demonstrated. To address this question, we used astrocyte-derived tumors (glioma) that lack functional Kir4.1 channels, and generated two glioma cell lines that stably express either AcGFP-tagged Kir4.1 channels or AcGFP vectors only. Kir4.1 expression confers the same K+ conductance to glioma membranes and a similar responsiveness to changes in [K+]o that characterizes differentiated astrocytes. Kir4.1 expression was sufficient to move the resting potential of gliomas from −50 to −80 mV. Importantly, Kir4.1 expression impaired cell growth by shifting a significant number of cells from the G2/M phase into the quiescent G0/G1 stage of the cell cycle. Furthermore, these effects could be nullified entirely if Kir4.1 channels were either pharmacologically inhibited by 100 μM BaCl2 or if cells were chronically depolarized by 20 mM KCl to the membrane voltage of growth competent glioma cells. These studies therefore demonstrate directly that Kir4.1 causes a membrane hyperpolarization that is sufficient to account for the growth attenuation, which in turn induces cell maturation characterized by a shift of the cells from G2/M into G0/G1. © 2007 Wiley-Liss, Inc.