A procedure is described which allows the simultaneous estimation of the rate of glucose influx from blood to brain and of the rate of glucose phosphorylation in discrete brain regions of the rat. The method is applicable over an experimental period of 5 min and involves sequential i.v. injections of [14C]2-deoxyglucose and [3H]2-deoxyglucose. Arterial blood samples are collected over the experimental period. After 5 min the rat is killed by microwave irradiation to the head and the brain dissected. The data obtained on glucose, 2-deoxyglucose and 2-deoxyglucosephosphate concentrations in plasma and brain are analyzed in terms of a model which relates the kinetics of 2-deoxyglucose uptake and phosphorylation to those of glucose. The model is based on measurements made of the relative rates of unidirectional influx of glucose and 2-deoxyglucose and of their relative fractional rates of phosphorylation.