NorBNI, GNTI, and JDTic are selective kappa opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14-21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists, naloxone or buprenorphine, prior to norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 one week later. Protection by a rapidly cleared reagent indicates that norBNI was not persisting at the site of action. In vitro binding of [3H]-U69,593 to KOR showed that Kd and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP, but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild-type mice, but not in KOR knockout mice. Pretreatment of mice with the JNK inhibitor SP600125 prior to norBNI partially reversed the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK.