RBE4 cells, an immortalized cell line derived from primary cultures of rat brain capillary endothelial cells, transport 3-O-methylglucose by a mechanism which is saturable with an apparent Km of 14.3 mM. The 3-O-MG uptake can be inhibited by other potential substrates and by non-competitive inhibitors, but not by L-glucose or sucrose. The transport is unaffected by removal or disruption of the sodium gradient. Under conditions of glucose deprivation the Vmax is increased by 100% with no significant change in Km. GLUT1 protein expression in glucose-deprived cells is also increased by 116%. This increase in Vmax can be blocked by the protein synthesis inhibitor, cycloheximide. Uptake was also increased in preconfluent cells and this increase was associated with increases in GLUT1 protein expression. This suggests that the changes in uptake are due to a de novo synthesis of transporters and not to insertion of transporters from an intracellular pool. This study would support the conclusion that the GLUT1 isoform is responsible for glucose transport in RBE4 cells and that these immortalized brain capillary endothelial cells may be used as an in vitro model for analysis of glucose transport regulation at the blood-brain barrier.