We studied the steady state and the regulation of intracellular Cl− activity (aCl−i) and the mechanisms of KCl uptake in cultured oligodendrocytes from mouse spinal cord using Cl−-selective microelectrodes. The majority of oligodendrocytes actively accumulated Cl− above passive distribution (2–3 mM), few cells showed a passive Cl− distribution. To identify the carriers mediating Cl− uptake, oligodendrocytes were maintained in a solution with low extracellular Cl− concentration ([Cl−]0) which resulted in a rapid decrease in aCl−i. The recovery of aCl−i above its passive distribution in normal [Cl−]0 was blocked in the absence of Na+ or in the presence of furosemide and of bumetanide, which has been reported to inhibit Na+/K+/Cl− cotransport. We therefore conclude that Cl− uptake is primarily due to the activity of a Na+/K+/Cl− transport system. Cl− uptake above passive distribution was not affected in HCO3−-free solution or in the presence of SITS and DIDS, indicating that Cl−/HCO3− exchange is not involved in Cl− uptake by oligodendrocytes. Elevation of [K+]0 induced an increase in aCl−i and, as shown earlier, intracellular K+ activity. This K+-induced Cl− uptake was not blocked by bumetanide, furosemide, SITS, or DIDS, suggesting that under conditions of raised [K+]0 the combined uptake of K+ and Cl− is not mediated by a carrier, but can be explained by the entry through channels driven by Donnan forces.