In order to study the long-term effects of a low extracellular glucose concentration on the metabolism and insulin production of the pancreatic B-cells a tissue culture system was applied in which isolated mouse pancreatic islets were maintained for 1 week in vitro at a glucose concentration of 0.6 mg/ml. Short-term experiments at the end of the culture period showed that both the oxygen uptake and the glucose oxidation were depressed but could be stimulated by increasing the glucose concentration of the incubation medium. Although incorporation of [3H]leucine into proinsulin and insulin was markedly depressed it could still be enhanced by increasing the glucose concentration of the incubation medium. By contrast, the insulin release could not be stimulated with glucose alone but with glucose plus theophylline a subnormal stimulation was achieved. The insulin response to glucose was, however, maintained when the glucose concentration of the culture medium was equal to or above 0.75 mg/ml. Similarly, the glucose sensitivity of the low-glucose cultured B-cells could be restored by raising the glucose concentration of the culture medium to 1.1 or 3.0 mg/ml during the last 2 days of culture. The results suggest that the insulin biosynthesis remains sensitive to glucose even when islets have been previously exposed to a low-glucose concentration for a prolonged period of time. The observations also support the view that the impaired insulin release in response to glucose observed during fasting reflects a lack of glycaemic stimulus to the B-cell.