Barbiturates are known to inhibit glucose transport mediated by the facilitative sugar transporter GLUTI. We have studied such inhibition in children with GLUT1-deficiency. Zero-trans influx of 14C-labeled 3-O-methyl glucose (3OMG) into erythrocytes of patients (n = 3) was 35% of controls (n = 6). Preincubation with 10 mM phenobarbital or pentobarbital reduced patients' 30MG influx to 17%. In patients and controls, preincubation with barbiturates significantly decreased Vmax in a dose-dependent manner (for pentobarbital, IC50 = 0.84 mM, patient 2). The apparent Km in individuals remained largely unchanged. Three-OMG influx without preincubation resulted in a stronger inhibition at lower barbiturate concentrations. The patients' data are discussed in the light of individual missense mutations (patient 1: R126L and K256V; patient 2: T310I; patient 3: S66F) in the GLUTI gene. In conclusion, in controls and patients with GLUT1-deficiency barbiturates interact with GLUT1, lowering its intrinsic activity. The use of barbiturates in this condition for anesthesia or as anticonvulsants could therefore potentially aggravate the existing glucose transport defect and may put these patients at increased risk.