There is evidence that depolarization of the pancreatic beta cell by glucose involves cell swelling and activation of the volume-regulated anion channel (VRAC). However, it is unclear whether cell swelling per se or accompanying changes in intracellular osmolality and/or ionic strength are responsible for VRAC activation. VRAC activity was measured in rat beta cells by conventional or perforated patch whole-cell recording. Cell volume was measured by video imaging. In conventional whole-cell recordings, VRAC activation was achieved by exposure of the cells to a hyposmotic bath solution, by application of positive pressure to the pipette, or by use of a hyperosmotic pipette solution. Increased concentrations of intracellular CsCl also caused channel activation, but with delayed kinetics. In perforated patch recordings, VRAC activation was induced by isosmotic addition of the permeable osmolytes urea, 3-O-methyl glucose, arginine, and NH4Cl. These effects were all accompanied by beta-cell swelling. It is concluded that increased cell volume, whether accompanied by raised intracellular osmolality or ionic strength, is a major determinant of VRAC activation in the beta cell. However, increased intracellular ionic strength markedly reduced the rate of VRAC activation. These findings are consistent with the hypothesis that the accumulation of glucose metabolites in the beta cell, and the resultant increase in cell volume, provides a signal coupling glucose metabolism with VRAC activation.