In standard 3H-opioid binding assays, the benzoylhydrazone derivative of naloxone (6-desoxy-6-benzoylhydrazido-N-allyl-14-hydroxydihydronormorphi none; NalBzoH) inhibited mu, kappa, and delta binding at nanomolar concentrations. At concentrations as low as 1 nM, it also produced a wash-resistant inhibition of opioid binding. [3H]NalBzoH binding typically gave a ratio of total to nonspecific binding of 8:1. Binding reached steady state levels by 1 hr and was linear with tissue concentration. [3H]NalBzoH labeled two classes of sites. The binding to one was easily reversible whereas the other was not and was termed pseudoirreversible. At 25 degrees, almost 90% of [3H]naloxone binding and approximately 60-75% of [3H]NalBzoH binding dissociated over 90 min. However, the remainder of [3H]NalBzoH binding, corresponding to pseudoirreversible binding, remained constant over the next 5 hr at 25 degrees and additional studies suggested a dissociation half-life of approximately 24 hr. Competition studies indicated that the reversible binding corresponded to neither mu nor delta binding and may represent a novel subtype of kappa receptor. Pseudoirreversible binding was predominantly to a combination of both mu 1 and mu 2 receptors. Despite its extremely slow rate of dissociation, pseudoirreversible binding was not covalent inasmuch as lowering the pH to 5 or adding the GTP analog 5'-guanylylimidodiphosphate [Gpp(NH)p] completely dissociated prebound [3H] NalBzoH. The ability of Gpp(NH)p to dissociate pseudoirreversible [3H]NalBzoH binding raised the possibility that the slow rate of dissociation was related to interactions with a guanine nucleotide-binding protein.