The effects of Na+, guanyl-5′-yl imidodiphosphate (Gpp(MH)p), and Mn2+ on the binding of dihydromorphine, ethylketocyclazocine, D-Ala2, D-Leu5- enkephalin, and diprenorphine to the opiate receptor were investigated. Three distinct binding sites, mu, delta, and kappa sites, were identified with the use of multiple tracer displacement curves. Moreover, this approach was used to determine the effects of Gpp (NH)p and metal ions on each individual binding site. At the mu and delta sites, Na+ and Gpp(NH)p each decreased and Mn2+ increased agonist binding affinities, with the exception of D-Ala2, D-Leu-enkephalin affinity which was not affected by Gpp(NH)p. None of these conditions markedly altered dihydromorphine and D-Ala2, Leu5-enkephalin binding to kappa sites, whereas the affinity of ethylke-tocyclazocine for kappa sites was decreased by Gpp(NH)p. Sodium ions lowered the capacity of mu sites and Gpp(NH)p reduced that of delta sites, while both agents increased the capacity of apparent kappa sites. These results demonstrate that each of the kinetically distinguishable binding sites is regulated differentially by metal ions and guanyl nucleotides. Simultaneous addition of Na+ and Gpp(NH)p greatly reduced the binding affinity of all three agonists at their respective high affinity sites (dihydromorphine at the mu site, D-Ala2, D-Leu5-enkephalin at the delta site, and ethylketocyclazocine at the kappa and mu sites). This result confirms previous observations that agonist binding is characterized by a large affinity reduction in the presence of both Na+ and guanyl nucleotides, and it extends this concept to each of the opiate receptor subtypes.