We investigated the role of ATP in the active efflux of doxorubicin (DOX) mediated by P-glycoprotein (P-gp), the multidrug-resistance (MDR) gene product, at the blood-brain barrier. In transient brain ischemic rats prepared with 4-vessel occlusion of vertebral and common carotid arteries for 20 min, a procedure that depleted their brain ATP content to 3% that of normal rats, the estimated permeability coefficient of DOX was increased 17-fold (to 243 ± 2.5 μL/min/g brain). When the ATP content recovered to a normal level by means of 30-min and 24-hr cerebral recirculation of blood, the permeability coefficient recovered to 14.0 ± 5.0 and 18.4 ± 2.3 μL/min/g brain (mean ± SEM, N = 3–6), respectively, very close to the control permeability (14.3 ± 1.5 μL/min/g brain). The uptake of DOX by primary cultured brain capillary endothelial cells expressing P-gp at the luminal membrane was increased significantly (up to 2-fold), which correlated well with the decrease of cellular ATP contents caused by treating the cells with metabolic inhibitors. Evidence for the ATP-dependent transport of DOX obtained from the present in vivo and in vitro studies strongly indicates that P-gp in the brain capillaries functions actively as an efflux pump in the physiological state, providing a major mechanism to restrict the transfer of DOX into the brain.