The K_ir4.1 channel is crucial for the maintenance of the resting membrane potential of glial cells, and it is believed to play a main role in the homeostasis of extracellular potassium. To understand its importance in these two phenomena, we have measuredin vivothe variations of extracellular potassium concentration ([K⁺]_o) (with potassium-sensitive microelectrodes) and membrane potential of glial cells (with sharp electrodes) during stimulations in wild-type (WT) mice and glial-conditional knock-out (cKO) K_ir4.1 mice. The conditional knockout was driven by the human glial fibrillary acidic protein promoter,gfa2. Experiments were performed in the hippocampus of anesthetized mice (postnatal days 17–24). Low level stimulation (<20 stimuli, 10 Hz) induced a moderated increase of [K⁺]_o(<2 mmincrease) in both WT and cKO mice. However, cKO mice exhibited slower recovery of [K⁺]_olevels. With long-lasting stimulation (300 stimuli, 10 Hz), [K⁺]_oin WT and cKO mice displayed characteristic ceiling level (>2 mmincrease) and recovery undershoot, with a more pronounced and prolonged undershoot in cKO mice. In addition, cKO glial cells were more depolarized, and, in contrast to those from WT mice, their membrane potential did not follow the stimulation-induced [K⁺]_ochanges, reflecting the loss of their high potassium permeability. Ourin vivoresults support the role of K_ir4.1 in setting the membrane potential of glial cells and its contribution to the glial potassium permeability. In addition, our data confirm the necessity of the K_ir4.1 channel for an efficient uptake of K⁺by glial cells.