In this study, we have examined glucose uptake and its regulation by insulin in primary cultured neurons. Glucose transport was assessed by measuring the initial rate of uptake of 3H-2-deoxyglucose, a glucose analog that is transported and phosphorylated but not further metabolized. The uptake of 2-deoxyglucose was saturable; measurements of the intracellular concentration of 2-deoxyglucose and 2-deoxyglucose-6-phosphate revealed that hexokinase activity rather than membrane transport is the rate-limiting step for glucose uptake. Insulin had no effect on 2-deoxyglucose uptake at low (0.2 mM) or high (20 mM) concentrations of substrate. The order of potency of other hexoses to competitively inhibit the accumulation of 2-deoxyglucose was D-glucose (0.2 mM) = D-mannose (0.2 mM) greater than 3-0-methylglucose (9 mM) greater than D-galactose (90 mM). Cytochalasin B was a potent inhibitor of 2-deoxyglucose uptake (IC50 = 500 nM) and phloretin was more potent than ploridzin in inhibiting uptake. The structure of glucose transporters was examined by photoaffinity labeling using 3H-cytochalasin B and by immunologic detection using antibodies raised against the human erythrocyte transporter. 3H-cytochalasin B labeled two proteins of 55 kDa and 43 kDa and the antibody recognized primarily a 43 kDa protein. The subcellular distribution of glucose transporters, estimated by measuring the number of specific cytochalasin B binding sites in subfractions of neuronal homogenates, showed 3.62 pmol/mg protein in the 11,000g pellet and 1.34 pmol/mg protein in the 200,000g pellet. CONCLUSIONS 1) Neuronal glucose transport is not acutely regulated by insulin. 2) The kinetics of 2-deoxyglucose uptake into neurons are determined largely by hexokinase activity rather than membrane transport. 3) The apparent molecular weight of neuronal glucose transporters is similar to transporters in other tissues. 4) The number of glucose transporters per milligram of protein is relatively low in neurons compared to other tissues.