The concept that cytosolic free calcium is the primary signal for insulin secretion is generally accepted, but studies with intact pancreatic beta-cells of the cytosolic free calcium concentration-insulin secretion relationship have produced contradictory and sometimes confusing data. We designed a superfusion system of a pancreatic beta-cell line, MIN6, loaded with fura-2, which allowed simultaneous measurement of cytosolic free calcium concentration and insulin secretion. MIN6 cells released insulin in response to high glucose, thus resembling events in normal islet cells. Cytosolic free calcium concentration and insulin secretion rapidly increased, and the increase was suppressed by mannoheptulose or by sodium azide. This increase was suppressed by lowering the temperature of the medium. Cytosolic free calcium concentration and the insulin secretion induced by leucine were not influenced by mannoheptulose but were inhibited by sodium azide. In RINm5F cells, cytosolic free calcium concentration and insulin release were slightly suppressed by glucose but were increased by ionomycin. There was a close relation between the rise in cytosolic free calcium concentration and insulin secretion in all cases. Our findings provide a direct evidence that a rise in cytosolic free calcium concentration depends on glucose metabolism and is a primary signal for insulin secretion.