We investigated the effect of 24 h of exposure to various glucose concentrations on insulin secretion by isolated rat pancreatic islets and purified rat β-cells. Compared with islets cultured with standard medium (5.5 mM glucose), islets cultured with 16.7 mM glucose showed a higher basal insulin release (means ± SE, 3.0 ± 0.5 vs. 0.7 ± 0.2%, n = 8, P < .005) and reduced glucose-stimulated insulin secretion (2.4 ± 0.3 vs. 5.8 ± 0.4%, n = 8, P < .005). Similar results were also obtained with purified β-cells. The effect of high glucose was time dependent (present after 12 h, maximal after 24 h) and reversible: when islets cultured with high glucose were transferred to standard medium, normal responsiveness to glucose was restored within 8 h and normal basal release within 24 h. Mannitol, 3-O-methylglucose, and 2-deoxyglucose were not able to mimic the effects of glucose. Islets or purified β-cells cultured in the presence of high glucose had a normal response when stimulated with glyburide, dibutyryl cyclic AMP, and isobutylmethylxanthine. Tunicamycin, an inhibitor of N-terminal glycosylation, prevented glucose-induced desensitization when added during 24 h of islet culture with 16.7 mM glucose. Swainsonine, another agent that influences glycosylation, had a similar effect. Our study indicates 1) that 24 h of exposure to high glucose induces a specific and reversible impairment of insulin secretion in response to glucose, 2) that this is a direct effect of glucose on β-cells, and 3) that islet glucose metabolism and glycosylation processes may play a critical role in determining glucose desensitization.