Repeated hypoglycemia increases the glycemic thresholds of responses of counterregulatory hormones and of symptoms to subsequent hypoglycemia. This may in part be due to cerebral adaptation to hypoglycemia, which involves glucose transporter-1 (GLUT1) and glucose transporter-3 (GLUT3). To investigate the role of brain GLUT1 and GLUT3 in cerebral adaptation to chronic hypoglycemia, GLUT1 and GLUT3 mRNA and protein expressions were determined in rat brain using RT-PCR and Western blot analyses after 4- and 8-day hypoglycemic insults. Hypoglycemia was induced in rats by twice daily subcutaneous injection of intermediate-acting insulin with dosage adjustment according to the blood glucose levels. Target level of hypoglycemia (< 2.5 mmol/l) was achieved at least once a day in all rats included. Control rats received saline injections. Blood glucose levels during the 4 and 8 days of insulin treatment were 2.18 +/- 0.12 and 2.68 +/- 0.07 mmol/l, respectively. Following the 4 and 8 days of hypoglycemia, GLUT1 mRNA levels did not significantly change. GLUT3 mRNA expressions after the 4 days of hypoglycemia increased by 36.9 +/- 9.4% compared with that in control rats (P = 0.031), but after the 8 days of hypoglycemia, did not change. On Western blot analysis of total particulate rat brain membrane, amount of 55-kDa isoform of GLUT1 protein did not change after 4- and 8-day hypoglycemia (88.1 +/- 4.9% of control, P = 0.240; 92.1 +/- 1.4% of control, P = 0.096, respectively). In contrast, the expression of GLUT3 protein in the 4-day hypoglycemic rats increased by 51.4 +/- 8.4% compared with that in control rats (P = 0.004). After the 8 days of hypoglycemia, the expression also tended to increase by 44.9 +/- 14.4% (P = 0.119). There was an inverse correlation between the amount of GLUT3 protein expression and mean blood glucose levels in 4-day hypoglycemic and control rats (r = -0.886, P = 0.019). These data suggest that GLUT3 isoform plays a role in the cerebral adaptation to chronic hypoglycemia.