Abstract Secretion of insulin from pancreatic the ß-cells and a mouse insulinoma cell line MIN6 with glucose stimulation results in an exocytotic reaction through membrane depolarization and an increase of intracellular Ca 2+ concentration ([Ca 2+] i). To investigate the relationship between membrane depolarization and intracellular calcium mobilization in a mouse insulinoma cell line, MIN6, we developed a simultaneous double-probe imaging video-microscope system, which could simultaneously detect two different kinds of fluorescent signals from a single cell. When MIN6 cells, which retain glucose-induced insulin secretion like pancreatic ß-cells, were stimulated by increasing the extracellular concentration of glucose from 5 to 25 mM, the membrane potential was quickly increased after the stimulation, but elevation of [Ca 2+] i was observed ca. 40 s after the increase in membrane potential. In the case of high K +-stimulation, such a time lag was not observed. When the extracellular concentration of glucose was increased stepwise from 5 to 25 mM, the number of cells which showed change of both membrane potential and [Ca 2+] i was increased, indicating that MIN6 cells show the so-called all-or-none-type of response. It was also found that cells which formed colonies responded to glucose stimulus. This method would be useful not only for investigating the detailed relationship between membrane depolarization and intracellular calcium mobilization in MIN6 cells as well as in the pancreatic ß-cells, but also for studying the signaling mechanisms in many kinds of cells.