Previously, demonstrated that GLUT2 mRNA and protein are increased in liver of streptozocin-induced diabetic rats. To examine the mechanisms whereby GLUT2 mRNA is regulated, we cultured isolated hepatocytes in the absence and presence of various concentrations of glucose. Culture of hepatocytes in high glucose concentration (27.8 mM) for 20 h induced a 3.2-fold increase in GLUT2 mRNA levels compared with hepatocytes cultured without D-glucose. Interestingly, D-mannose and D-fructose could substitute for D-glucose to elevate the GLUT2 mRNA level, whereas 3-O-methyl-D-glucose, 2-deoxy-D-glucose, and sucrose, which were not metabolized or taken up by the cells, were without effect. Insulin had no significant effect on GLUT2 mRNA levels in hepatocytes in the presence or absence of D-glucose. Therefore, the regulation of the GLUT2 gene by D-glucose in hepatocytes is contrary to that reported for GLUT1 and GLUT4 genes, which are downregulated by D-glucose. These results also suggest that the elevated GLUT2 mRNA level observed in diabetic rat liver is due to the high blood glucose concentration rather than to insulin deficiency.