Nor-binaltorphimine (nor-BNI) is kappa opioid receptor (KOR) antagonist with the extremely long duration in mice analgesic assay, in vivo. For the evaluation of long-lasting effect of nor-BNI, brain content and serum concentration of nor-BNI were quantified in comparison with those of naloxone (a short-acting non-specific opioid receptor antagonist) by high-performance liquid chromatography with electrochemical detector in mice. After concomitant administration (20mg/kg, s.c.) of nor-BNI and naloxone, nor-BNI in brain and serum showed biphasic elimination, with a rapid phase for 0.75-4 hr and a slow phase for 4 - 48 hr. Elimination rate in brain was slower than that of serum. Naloxone in brain and serum was detected for 3 hr and 4 hr, respectively. The brain/serum ratio of nor-BNI gradually increased over 0.75 - 48 hr, while that of naloxone rapidly declined. After concomitant administration (30mg/kg, s.c.) of nor-BNI and naloxone, brain nor-BNI was detected in all mice from day 1 to day 21 and in two of six mice at day 28, while serum nor-BNI was detected in all mice at day 1, three of seven at day 3 and one of six at day 7. After that, serum nor-BNI was not detected. Naloxone in brain and serum was not detected at day 1. These results provide pharmacokinetic support for the long-lasting antagonistic effects of nor-BNI.