Tissue aggregation and exocrine contamination are problems encountered in gradient separation of pancreatic islets. Here we report that sorbitol used as an osmotic component in Percoll gradients gives a low ionic strength gradient with improved purity of islet fraction, less islet aggregation and reduced time for final manual rinsing following separation in gradients with NaCl as osmotic component. Previous reports have indicated that long-term (weeks) exposure to high sorbitol concentrations leads to low intracellular levels of inositol phosphates and subsequent effects on the intracellular signal transduction in cells. In our model, short-term exposure to high sorbitol concentrations had no effect on the accumulation of the inositol phosphates or insulin secretion caused by glucose. On the other hand, sorbitol increased the basal insulin secretion threefold, apparently via a non-stimulatory mechanism. Therefore, we conclude that sorbitol is preferable to NaCl as the osmotic component in Percoll gradient separation of rat pancreatic islets, although long-term exposure should be avoided due to potential toxic effects.