Regulation of the volume sensitivity of the swelling-activated organic osmolyte/anion channel VSOAC by intracellular electrolytes was examined in intact and patch-clamped C6 glioma cells. In intact cells, VSOAC activation was monitored by [3H]taurine efflux measurements, and intracellular electrolyte concentrations were manipulated by acclimation to hypertonic medium for varying periods of time. Hypertonic shrinkage was followed by a rapid and complete regulatory volume increase mediated by electrolyte accumulation that elevated intracellular Na+, K+, and Cl- concentrations. During prolonged (4-48 h) exposure to hypertonicity, electrolyte concentrations decreased gradually as cells accumulated the organic osmolyte myo-inositol. VSOAC activation induced by cell swelling of 35-40% increased as a function of the time of exposure to hypertonicity and was inversely correlated with measured intracellular Na+, K+ and Cl- levels. In patch-clamped cells, swelling-induced Cl- current activation was unaffected by acclimation conditions but was inhibited by increasing the concentration of electrolytes in the patch pipette solution. Quantification of the relationship between VSOAC activation and cell swelling demonstrated that increases in intracellular electrolyte levels increase VSOAC volume set point. Regulation of VSOAC volume sensitivity by electrolytes allows cells to selectively utilize electrolytes or a combination of electrolytes and organic osmolytes for regulatory volume decrease (RVD). Control over the type of solute used for volume regulation is advantageous, allowing cells to control intracellular ionic composition and prevent increases in cytoplasmic ionic strength during RVD.