Receptor binding properties of the naturally occurring opioid heptapeptide MERF were studied in rat brain membrane preparations using tritium-labeled derivative of the peptide with 40 Ci/mmol specific radioactivity. Binding assays were performed in the presence of broad-spectrum peptidase inhibitors at 0°C. Under these conditions, the equilibrium binding was achieved in 30-40 min, and approximately 90% of the applied radioligand remained unchanged as determined by HPLC analysis. The apparent affinity (K d value) of [ 3 H]Met-enkephalin-Arg 6 -Phe 7 , calculated from saturation binding data, was 10.2 6 2.5 nM, and the maximal number (B max ) of the heptapeptide binding sites was found to be 468 6 43 fmol/mg protein. About half the sites represent nonopioid sites because the B max was only 255 6 30 fmol/mg, when the nonspecific binding was measured with 1 mM naloxone. The rank order potencies of the examined compounds revealed that the opioid component of [ 3 H]Metenkephalin-Arg 6 -Phe 7 recognition sites are probably not m and certainly not k 1 sites, whereas these sites are characterized by a k 2 -like binding profile. Considering the discrepancies between rat and frog brain found in the affinity of some compounds, including naltrindole and norbinaltorphimine, the presence of a novel, MERF-selective ''heptapeptide'' binding site in rat brain membranes is also suggested. A number of the heterologous competition curves could be described by a high-affinity stereospecific component and a substantially lower-affinity binding element, which could completely be displaced with several peptide ligands such as Met 5 -enkephalin, dynorphin (1-13) , and unlabeled MERF but not by other compounds such as [D-Ala 2 -(Me)Phe 4 -Gly 5 -ol]enkephalin, morphine, or naloxone. [ 3 H] Met-enkephalin-Arg 6 -Phe 7 binding can also be inhibited by FMRFamide analogs and sigma receptor ligands, such as (1)N-allyl-normetazocine and haloperidol, although with moderate affinity. It is concluded that the stereospecific high-affinity binding is of opioid in character, whereas the residual sites characterized with their lower affinity are naloxone-insensitive nonopioid sites.