In light of more recent knowledge concerning endogenous opioid peptides and their multiple opiate receptors, we reevaluated the effects of morphine tolerance on opiate receptor binding parameters. Rats were implanted with morphine or placebo pellets, and [3H][D-Ala2,D-Leu5]enkephalin ([3H]DADL) was used to label brain membranes. Utilizing the technique of binding surface analysis, we observed a selective 47% up-regulation of lower affinity [3H]DADL binding sites (mu-noncompetitive delta binding sites) in morphine pelleted rats. To corroborate these results, we treated brain membranes with the site directed alkylating agent FIT (N-phenyl-N-[1-(2-p-isothiocyanato)phenyl-ethyl)-4-piperidinyl] propanamide), which results in membranes highly enriched with the lower affinity [3H]DADL binding site. Scatchard plots of [3H]DADL binding to FIT-treated membranes also revealed that chronic morphine treatment produced a 60-65% up-regulation of the mu-noncompetitive delta binding site. These data indicate that chronic morphine alters a selective subpopulation of opiate receptors that may play a role in the mechanisms of opiate tolerance and physical dependence.