cAMP is an important messenger in the regulation of insulin and glucagon secretion. The aim of the present study was to investigate the effects of glucose and adenylyl cyclase-activating hormones on cAMP in a- and b-cells within their natural islet environment. Islets from C57/Bl mice were infected with adenovirus expressing a fluorescent translocation biosensor reporting cAMP concentration beneath the plasma membrane ([cAMP] pm). Changes in biosensor localization were recorded from the superficial islet cells adhering to the coverslip using ratiometric evanescent wave microscopy. b-cells were identified based on their large size and [cAMP] pm-lowering in response to adrenaline, while a-cells were smaller and reacted to adrenaline with increase of [cAMP]pm. In the presence of 3 mM glucose [cAMP]pm was low and stable. Elevation of the glucose concentration to 11, 20 or 30 mM induced a marked increase of [cAMP]pm with slow oscillations (0.13–0.59/min) both in a- and b-cells. The oscillations persisted with diminished amplitudes after removal of extracellular Ca²⁺. Glucagon-like peptide 1 and glucagon (1–100 nM) induced prompt [cAMP]pm elevation with oscillations in both a- and b- cells exposed to 3 mM glucose. The duration of each [cAMP]pm oscillation increased dose- dependently with hormone concentration and in b-cells the amplitudes were amplified by increasing the glucose concentration to 11 mM. It is concluded that glucagon, GLP-1 and glucose induce pronounced oscillations of [cAMP]pm in individual a- and b-cells within intact pancreatic islets. Such oscillations should contribute to the pulsatile release of glucagon and insulin. The glucose- induced elevation of [cAMP]pm in a cells may underlie a recently identified stimulatory action of the sugar on glucagon secretion.