This study examined the effect of glucose and 12-O-tetradecanoylphorbol-13-acetate (TPA) on insulin secretion in isolated human insulinoma cells. In addition, we analyzed conventional PKC alpha and beta activation in the membrane fractions, respectively. Treatment with 5 mM and 20 mM glucose for 5 min and 20 min resulted in 6-7-fold increases in insulin secretion, and treatment with 1 microM TPA for 5 min also resulted in 3-fold increases in insulin secretion from the basal level. Immunoblot analysis of membrane fractions showed increases in PKC alpha and beta immunoreactivities after treatment with 5 mM, 20 mM glucose and 1 microM TPA. Translocations of PKC alpha after treatment with glucose and TPA were greater than those of PKC beta in membrane fractions. These results suggest that TPA independently provokes insulin secretion via PKC activation and that PKC alpha and beta activation may be involved in insulin secretion in human insulinoma cells.