A decrease in the expression of inwardly rectifying potassium (Kir) currents is a characteristic feature of retinal glial (Muller) cells in various retinopathies, e.g., after transient retinal ischemia. We used short-term retinal organ cultures to investigate whether similar physiological alterations can be induced under in vitro conditions. During 4 days in vitro, Muller cells displayed a decrease in Kir currents and an increase in transient A-type potassium currents which was similar to the alterations in membrane physiology during ischemia-reperfusion in vivo. In addition, gliosis of Muller cells both in vivo and in organ cultures was associated with cellular hypertrophy and an alteration in osmotic swelling characteristics. Whereas Muller cells in control retinae did not swell under hypotonic stress, cells in postischemic retinae and in organ cultures swelled upon hypotonic challenge. Therefore, Muller cells in organ cultures can be used to investigate distinct aspects of ischemia-induced Muller cell gliosis. Both the decrease in Kir currents and the alteration in osmotic swelling may reflect a dysfunction of Muller cells regarding the control of the ionic and osmotic homeostasis in the retina.