Capsaicin inhibited the equilibrium specific binding of endogenous opioid-like peptide ligands such as endomorphin-1, nociceptin, and dynorphin((1-17)) in rat brain membrane preparations. We studied the in vitro effect of capsaicin (1-10 microM) on homologous and heterologous competitive binding of opioid ligands, using unlabeled synthetic peptides and the following tritiated compounds: [(3)H]endomorphin-1, [(3)H]endomorphin-2, [(3)H]nociceptin((1-17)) and [(3)H]dynorphin((1-17)). Capsaicin-dependent inhibition was also observed in [(35)S]GTPgammaS stimulation assays in the presence of certain opioid peptides. The inhibition of opioid binding was further investigated using other synthetic and natural mu-opioid ligands such as [D-Ala(2),(NMe)Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and naloxone. The decrease in opioid ligand affinity upon capsaicin treatments was most apparent with endomorphin-1, followed by nociceptin and dynorphin. The binding of other investigated opioids were not affected in the presence of capsaicin. In [(3)H]endomorphin-1 binding assays, capsazepine antagonized the inhibitory effect of capsaicin in rat brain membranes suggesting the involvement of TRPV1 receptors. In Chinese hamster ovary (CHO) cells stably expressing mu-opioid receptors, but lacking vanilloid receptors, the inhibition by capsaicin on the binding of [(3)H]endomorphin-1 was not present. It is concluded that the inhibitory effect of capsaicin on the receptor binding affinity of endogenous opioid peptides in brain membrane preparations seems not to be a direct effect, it is rather a negative feedback interaction with opioid receptors.