Barbiturates reduce cerebral blood flow, metabolism, and Glc transfer across the blood-brain barrier. The effect of barbiturates on hexose transport in cultured mammalian cell lines and human erythrocytes was studied. Pentobarbital inhibits [3H]-2-dGlc uptake in 3T3-C2 murine fibroblasts by approximately 95% and approximately 50% at 10 and 0.5 mM, respectively. Uptake of [3H]-2-dGlc is linear with time in the presence or absence of pentobarbital, and the percent inhibition is constant. This suggests that hexose transport, not phosphorylation, is inhibited by barbiturates. Inhibition by pentobarbital of hexose transport in 3T3-C2 cells is rapid (< 1 min), is not readily reversible, is not altered by the presence of albumin [1% (w/v)], and is independent of temperature (4-37 degrees C) and the level of cell surface GLUT-1. The IC50's for inhibition of hexose transport in 3T3-C2 cells by pentobarbital, thiobutabarbital, and barbital are 0.8, 1.0, and 4 mM, respectively. This is consistent with both the Meyer-Overton rule and the pharmacology of barbiturates. Neither halothane (< or = 10 mM) nor ethanol [< or = 0.4% (v/v)] significantly inhibits hexose transport. Inhibition by pentobarbital (0.5 mM) of [3H]-2-dGlc uptake by 3T3-C2 cells decreases the apparent Vmax (approximately 50%) but does not alter the apparent Km (approximately 0.5 mM). Inhibition of hexose transport by barbiturates, but not ethanol [< or = 0.4% (v/v)], is also observed in human erythrocytes and four other cultured mammalian cell lines. Pentobarbital quenches (Qmax approximately 75%) the intrinsic fluorescence of purified and reconstituted GLUT-1 (Kd approximately 3 mM). Quenching is independent of Glc occupancy, is unchanged by mild proteolytic inactivation, and does not appear to directly involve perturbations of the lipid bilayer. We propose that barbiturates can interact directly with GLUT-1 and inhibit the intrinsic activity of the carrier. Glc crosses the blood-brain barrier primarily via the GLUT-1 of the endothelial cells of cerebral capillaries. Partial inhibition of this process by barbiturates may be of significance to cerebral protection.