Insulin is believed to increase glucose permeability of adipocytes by regulating the incorporation of glucose transporters into the plasma membrane by exocytosis. This process involves fusion of membrane-bound cellular compartments with the plasma membrane, thus influencing the plasma membrane area. However, insulin-induced changes in plasma membrane area have not yet been demonstrated. In the present study we monitored fluorescence intensity with a confocal microscope to study the effect of insulin on adipocyte plasma membrane area. After cell isolation and adhesion to a glass cover-slip, adipocytes were stained with the dye FM1-43, a membrane area reporter. At rest, the rate of fluorescence intensity increase was initially high, but gradually stabilized at 2%/min. This steady increase in fluorescence is due to a slow rate of exocytosis coupled to endocytosis, since the removal of FM1-43 from the bath did not abolish FM1-43 fluorescence. Insulin addition caused an abrupt increase of fluorescence intensity of 4%/min, which was significantly higher than in controls. These results suggest rapid, insulin-induced incorporation of new membrane into the plasma membrane by exocytosis.