AbstractIn glial cells, inwardly rectifying K+ channels (Kir) control extracellular [K+]o homeostasis by uptake of K+ from the extracellular space and release of K+ into the microvasculature. Kir channels were also recently implicated in K+‐associated water influx and cell swelling. We studied the time‐dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co‐localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time‐lapse‐two‐photon laser‐scanning microscopy in situ. Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co‐application of hypotonic solution and Ba2+, a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1−/− mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co‐application with Ba2+. Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.