The effects of glucose and tolbutamide on the sodium handling of the pancreatic beta-cells were evaluated by measuring the total sodium content in intact islets from ob/ob-mice by integrating flame photometry and the free ion in individual beta-cells by dual wavelength fluorometry. Whereas increasing the glucose concentration from 3 to 20 mM resulted in a lowering of sodium, the addition of 100 microM tolbutamide caused a rise. The above-mentioned effects were most marked (about 50%) for the physiologically significant free sodium. The data indicate a more important role for Na+ in the regulation of insulin release than so far acknowledged. Increase of Na+ may contribute to the secretory response to hypoglycemic sulfonylureas by providing an additional rise of cytoplasmic Ca2+.