Volume changes and the whole cell ionic currents activated by a gradual osmolarity reduction (GOR) of -1.8 mOsm/min, were characterized in C6 glioma cells. Cells swell less in this condition than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca²⁺-dependent. Whole cell currents and separate Cl^- and K⁺ currents were recorded in nominally intracellular Ca²⁺-free conditions or in the presence of 200 nM intracellular Ca²⁺ in the patch pipette. In Ca²⁺-free media, GOR activated predominantly whole cell outward currents. Cells increasingly depolarized from the initial -79mV to reach a steady state of -54 mV when external osmolarity decreased 18% (H-18%). Separated recordings of Cl^- and K⁺ currents showed the activation at H-3%, of an outwardly rectifying Cl- current, with conductance of 1.6 ± 0.18 nS, sensitive to niflumic acid and NPPB, and followed by an outwardly rectifying K⁺ current activated at H-18%, with conductance of 4.1 ± 0.47 nS, which is blocked by clofilium but insensitive to TEA, Ba²⁺, 4AP or quinidine. In the presence of 200 nM Ca²⁺ in the patch pipette, predominantly outward whole cell currents activate at H-3%, without a change in membrane potential, until H-13% when cells depolarized from -77 to -63 mV. Under these intracellular Ca²⁺ conditions, a K⁺ current activates as early as H-1% showing a rapid increase in conductance. The Ca²⁺-dependent K⁺ current is suppressed by charybdotoxin and insensitive to clofilium. These results support the activation of two different K⁺ channels in response to GOR in the same type of cell. These K⁺ channels activate by different signals, i.e. Ca²⁺ and osmolarity, and showed different osmolarity thresholds for activation. The maximal value of total currents was consistently higher in the presence of Ca²⁺, particularly at small osmolarity reductions, reflecting the contribution of the Ca²⁺-activated K⁺ current. The efflux of taurine and glutamate, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39% and H-33%, respectively. This observation clearly separates the Cl^- and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions