This study shows that nucleotides, as well as ions, regulate the opiate receptors of brain. GMP-P(NH)P and Na(+) reduce the amount of steady-state specific [(3)H]dihydromorphine binding and increase the rate of dissociation of the ligand from the opiate receptor. In contrast, Mn(2+) decreases the rate of ligand dissociation and antagonizes the ability of Na(+) to increase dissociation. The effects of GMP-P(NH)P on steady-state binding and dissociation are not reversed by washing. Only GTP, GDP, ITP, and IMP-P(NH)P, in addition to GMP-P(NH)P, increase the rate of dihydromorphine dissociation. The site of nucleotide action appears to have high affinity: <1 muM GMP-P(NH)P produces half-maximal increases in ligand dissociation. GMP-P(NH)P- and Na(+)-directed increases in dissociation have also been found for the opiate agonists [(3)H]etorphine, [(3)H]Leu-enkephalin, and [(3)H]Met-enkephalin and the opiate antagonist [(3)H]naltrexone. Mn(2+)-directed decreases in dissociation have been found for the agonist [(3)H]-etorphine and the antagonist [(3)H]naltrexone. Although the plasma membrane receptors for a number of other neuro-transmitters and hormones are also regulated by guanine nucleotides, the opiate receptors appear unique because only they show nucleotide regulation of both agonist and antagonist binding.