A classical action of glucocorticoids (GCs) is to inhibit glucose uptake into various peripheral tissues. Two recent reports suggest that GCs do the same in the brain. Because of the in vivo nature of those studies, it was impossible to determine whether this inhibition occurred at the blood-brain barrier, and/or within neurons and glia themselves. In order to answer this and other mechanistic questions, we examined the effects of GCs on glucose transport in primary brain cultures. We established that uptake of 14C-2-deoxyglucose into hippocampal cultures was linear over a 15-min period and was inhibited by D-glucose and the uptake inhibitor cytochalasin B. Using this system, we found the following. (1) Both corticosterone and dexamethasone inhibited uptake into cultures containing both neurons and glia. (2) The effect was dose-dependent; steroid concentrations in the nanomolar range inhibited uptake from 20 to 33%. The effect was time-dependent, with more than 4 h of steroid exposure needed for inhibition. (3) Non-GC steroids did not inhibit uptake. (4) The GC inhibition seemed to be mediated by the type II (glucocorticoid) corticosteroid receptor. The effect was blocked by a type II, but not a type I (mineralocorticoid) receptor antagonist. Moreover, corticosterone inhibited only at concentrations well above the Kd for the type I receptor. Finally, aldosterone inhibited transport when applied at concentrations that bound heavily to type II receptors. (5) Corticosterone did not inhibit uptake in hypothalamic, cerebellar or cortical cultures, despite the presence of corticosteroid receptors in these cultures. (6) GCs inhibited uptake in both neuron- and glia-enriched hippocampal cultures.