This study examines the effects of chronically elevated glucose levels on the survival and function of purified rat β-cells. Prolonged exposure (9 days) of β-cell aggregates to 20 mmol/l glucose did not lead to cell losses, but reduced the amount of insulin secreted in response to glucose. This decrease was not caused by cellular desensitization but resulted from the lower cellular insulin content after a prolonged imbalance between stimulated rates of insulin synthesis and release. Virtually all β-cells exhibited a state of metabolic and biosynthetic activation, which was maintained for at least 2 h in glucose-depleted media. Their rates of protein and insulin synthesis were amplified by glucose, reaching (half-) maximal stimulation at lower glucose concentrations (2 and 5 mmol/l, respectively) than control cells cultured at 10 mmol/l glucose (5 and 10 mmol/l, respectively). As for insulin release, the net glucose effect on insulin synthesis was markedly reduced as compared with that in control cells. This was also the case after culture at 6 mmol/l glucose. In the latter condition, the lower glucose-inducible activities were caused by cellular desensitization, with 50% of the β-cells unresponsive to glucose and the other 50% responding with a lower sensitivity (half-maximal stimulation at 7 mmol/l glucose). Comparison of β-cells cultured at the three glucose concentrations indicated that prolonged exposure to elevated glucose levels increases the number of degranulated cells, of cells with a high proportion of immature insulin granules, and of cells with glycogen deposition-morphologic features previously described in conditions of hyperglycemia. It is concluded that chronic exposure (9 days) of rat β-cells to elevated glucose levels induces a prolonged state of β-cell activation and glucose hypersensitivity rather than a glucotoxicity or glucose desensitization. This shift in the functional state of the β-cell population is responsible for a reduced insulin release in response to glucose, as observed in other conditions of prolonged exposure to high glucose levels.