The blood-brain barrier (BBB) transport of naloxone, a potent and specific opioid antagonist, was investigated in rats using the brain uptake index method and the brain efflux index method. The apparent influx clearance of [ 3 H]naloxone across the BBB was 0.305 mL/min/g brain. [ 3 H]naloxone was eliminated from the brain with an apparent elimination half-life of 15.1 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [ 3 H]naloxone across the BBB was 0.152 mL/min/g brain, which was calculated from the elimination rate constant (4.79 Â 10 À2 min À1 ) and the distribution volume in the brain (3.18 mL/g brain). The influx clearance across the BBB was two times greater than the efflux clearance. The elimination of [ 3 H]naloxone from the brain was not inhibited in the presence of the typical P-glycoprotein (P-gp) inhibitors such as quinidine, verapamil, vinblastine, and vincristine, indicating that naloxone is not a P-gp substrate in the rat. In vitro experiments by using human multidrug resistance 1 (MDR1)/P-gp overexpressing HeLa cells showed that the uptake of naloxone by the cells did not change in the presence of the P-gp inhibitors. In conclusion, the present results obtained from in vivo and in vitro studies suggest that P-gp is not involved in the BBB transport of naloxone.