We used the db/db mouse to determine the nature of the secretory defect in intact islets. Glucose tolerance was compared in db/db and wild-type (WT) mice. Isolated islets were used: to measure insulin secretion and calcium in a two-photon assay of single-insulin-granule fusion; and for immunofluorescence of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs). The 13-18-week-old db/db mice showed a diabetic phenotype. Isolated db/db islets showed a 77% reduction in insulin secretion induced by 15 mmol/l glucose and reductions in the amplitude and rise-time of the calcium response to glucose. Ionomycin-induced insulin secretion in WT but not db/db islets. Immunofluorescence showed an increase in the levels of the SNAREs synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) in db/db islets, but reduced syntaxin-1A. Therefore, db/db islets have both a compromised calcium response to glucose and a compromised secretory response to calcium. Two-photon microscopy of isolated islets determined the number and distribution of insulin granule exocytic events. Compared with WT, db/db islets showed far fewer exocytic events (an 83% decline at 15 mmol/l glucose). This decline was due to a 73% loss of responding cells and, in the remaining responsive cells, a 50% loss of exocytic responses per cell. An assay measuring granule re-acidification showed evidence for more recaptured granules in db/db islets compared with WT. We showed that db/db islets had a reduced calcium response to glucose and a reduction in syntaxin-1A. Within the db/db islets, changes were manifest as both a reduction in responding cells and a reduction in fusing insulin granules per cell.