Chronic hyperglycemia desensitizes beta-cells to glucose. To further define the mechanisms behind desensitization and the role of overstimulation, we tested human pancreatic islets for the effects of long-term elevated glucose levels on cytoplasmic free Ca2+ concentration ([Ca2+]i) and its relationship to overstimulation. Islets were cultured for 48 h with 5.5 or 27 mmol/l glucose. Culture with 27 mmol/l glucose obliterated postculture insulin responses to 27 mmol/l glucose. This desensitization was specific for glucose versus arginine. Desensitization was accompanied by three major [Ca2+]i abnormalities: 1) elevated basal [Ca2+]i, 2) loss of a glucose-induced rise in [Ca2+]i, and 3) perturbations of oscillatory activity with a decrease in glucose-induced slow oscillations (0.2-0.5 min(-1)). Coculture with 0.3 mmol/l diazoxide was performed to probe the role of overstimulation. Neither glucose nor diazoxide affected islet glucose utilization or oxidation. Coculture with diazoxide and 27 mmol/l glucose significantly (P < 0.05) restored postculture insulin responses to glucose and lowered basal [Ca2+]i and normalized glucose-induced oscillatory activity. However, diazoxide completely failed to revive an increase in [Ca2+]i during postculture glucose stimulation. In conclusion, desensitization of glucose-induced insulin secretion in human pancreatic islets is induced in parallel with major glucose-specific [Ca2+]i abnormalities. Overstimulation is an important but not exclusive factor behind [Ca2+]i abnormalities.