The effects of prolonged culture of rat pancreatic islets at a high glucose concentration were investigated. Isolated islets were cultured for either 1 day, 1 wk or 4 wk in 5.5 mM or 55 mM D-glucose or 5.5 mM D-glucose plus 49.5 mM L-glucose. At the end of the culture periods both the basal and stimulated rates of (pro) insulin and total protein synthesis were measured in short-term incubations and the islet insulin content was also determined. After prolonged culture (1-4 wk) the insulin content of islets maintained at 5.5 mM D-glucose (with or without L-glucose) increased 2.5-4 times, while that of the high-glucose exposed islets remained essentially unchanged. The (pro) insulin biosynthetic response to an acute glucose challenge, after both 1 day and 1 wk was less marked in the 55 mM than in the 5.5 mM glucose cultured islets. This may largely be due to the high basal rates of (pro) insulin biosynthesis observed in the high glucose cultured islets. At the end of the 4th culture week the rates of (pro) insulin biosynthesis were similar in all 3 experimental groups. Prolonged in vitro exposure of islets to an abnormally high glucose concentration does not result in any specific injurious effects on the .beta.-cells.