Naloxone benzoylhydrazone (NalBzoH) is a novel mixed agonist/antagonist. Against mu agonists, NalBzoH is a potent antagonist with a prolonged duration of action corresponding to its extremely slow rate of dissociation from mu receptors in binding assays. In the present studies, NalBzoH also antagonized mu analgesia, reversing both mu 1 and mu 2 analgesia independently elicited by intracerebroventricular or intrathecal [D-Ala2,MePhe4,-Gly(ol)5]enkephalin injections. It also antagonized kappa 1 analgesia elicited by U50,488H, and delta analgesia produced by intrathecal [D-Pen2,D-Pen5]enkephalin. Yet, at higher doses, NalBzoH alone produced analgesia in the tail-flick, hot plate and writhing assays. Neither the mu-selective antagonist beta-funaltrexamine, the delta-selective antagonist naltrindole, nor the kappa 1-selective antagonist norbinaltorphimine reversed NalBzoH analgesia in the tail-flick test. Analgesia observed with systemically administered NalBzoH was reversed easily by the antagonist WIN44,441 when it was given intracerebroventricularly, but not intrathecally. These observations confirm the opioid nature of NalBzoH analgesia and imply a supraspinal mechanism of action. In contrast, intrathecal, but not intracerebroventricular WIN44,441 reversed analgesia from systemic U50,488H quite potently. Thus, NalBzoH antagonizes mu, delta and kappa 1 actions while retaining its ability to elicit analgesia through a novel and distinct supraspinal kappa 3 system.