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Glucose and beta-hydroxybutyrate metabolism were compared in isolated cerebral microvessels from chronically diabetic and hypoglycemic rats. As noted previously, glucose oxidation and conversion to lactate are diminished in rats with streptozotocin-induced diabetes. The decrease in glucose metabolism did not result from selective damage to diabetic vessels during isolation, since the ATP level and the ATP/ADP ratio were similar to those of nondiabetic rats, and O2 consumption was increased. In addition, cerebral microvessel oxidation of beta-hydroxybutyrate was enhanced by diabetes. By contrast, microvessels from rats made chronically hypoglycemic by insulinoma engrafting 30 days earlier had a more than twofold increase in glucose oxidation and conversion to lactate, whereas their oxidation of beta-hydroxybutyrate was diminished by 50%. Unlike the insulinoma rats, no consistent increase in glucose metabolism was observed in microvessels from rats made hypoglycemic either by acute insulin administration or by a 4-day infusion of insulin. These results indicate that diabetes, and under some circumstances chronic hypoglycemia, markedly alters fuel metabolism in the cerebral microvasculature.