Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (Tmax) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible MichaelisMenten model, which is implicit to the large iso-inhibition constant (Kii) relative to physiological brain glucose content, we found that the apparent affinity constant (Kt) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both Tmax and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.