Palmitoylation of GLUT1 was investigated in brain capillaries. The glucose transporter was shown to be palmitoylated using [3H]palmitate labeling and immunoprecipitation. The labeling was sensitive to methanolic KOH or hydroxylamine hydrolysis, indicating the presence of an ester or thioester bond. The released fatty acid was analyzed by reverse-phase HPLC and was identified as [3H]palmitate. Specificity of the immunoprecipitation was assessed by competitive inhibition of anti-GLUT1 binding with a synthetic C-terminal peptide against which the antibody was raised. In vivo studies were performed using capillaries isolated from control rats, streptozotocin-induced diabetic rats and diet-induced hyperglycemic rats. Glycemia was increased 2- and 5-fold in the hyperglycemic and diabetic groups, respectively. GLUT1 expression was evaluated in the three groups by Western blot analysis. A 36% decrease in GLUT1 expression was observed in the diabetic group, while there was no significant variation in GLUT1 expression in the hyperglycemic group. Palmitoylation of GLUT1 was increased in both diet-induced hyperglycemic and diabetic groups. These results suggest that palmitoylation may be involved in the regulation of glucose transport activity in hyperglycemia.