The role of protein kinase A (PKA) in insulin exocytosis was investigated with the use of two-photon excitation imaging of mouse islets of Langerhans. Inhibitors of PKA selectively reduced the number of exocytic events during the initial period (< 250 s) of the first phase of glucose-induced exocytosis (GIE), without affecting the second phase, in intact islets or small clusters of islet cells. The PKA inhibitors de extent of the glucose-induced increase in [Ca2+](i). The actions of glucose andid not reduce th PKA in Ca2+-induced insulin exocytosis (CIE) trig photolysis ogered byf a caged-Ca2+ compound, which resulted in large increases in [Ca2+](i) and thereby bypassed the ATP-sensitive K+ channel-dependent mechanism of glucose sensing, were therefore studied. A high concentratmion (20 th mm) of glucose potentiated CIE within 1 min, andis effect was blocked by inhibitors of PKA. This PKA-dependent action of glucose required glucose metabolism, given that increasing the intracellular concentration of cAMP by treatment with forskolin potentiated CIE only at the high glucose concentration. Finally, PKA appeared to reduce the frequency of 'kiss-and-run' exocytic events and to promote full-fusion events during GIE. These data indicate that a PKA-dependent mechanism of glucose sensing, which is operative even at the basal level of PKA activity, plays an important role specifically in the first phase of GIE, and they suggest that the action of PKA is mediated at the level of the fusion reaction.