Specific receptors for the octapeptide FLFQPQRFamide (NPFF), a mammalian FMRFamide-like neuropeptide with anti-opiate properties have been identified in rat central nervous system. However, exploration of the biological role of this peptide requires a peptidase-resistant agonist. In this study, the stability and binding characteristics of [125I]DYLMeFQPQRFamide, a radioiodinated analogue of NPFF, on rat spinal cord tissue were determined and compared with those of [125I]YLFQPQRFamide, the reference ligand which previously permitted to characterize NPFF binding sites. In a binding assay, [125I]DYLMeFQPQRFamide remained intact in the presence of membranes without peptidase inhibitors, whereas [125I]YLFQPQRFamide was completely hydrolysed. The specific binding was time-dependent, dose-dependent, saturable and reversible. [125I]DYLMeFQPQRFamide shared the same binding characteristics as [125I]YLFQPQRFamide (Kd = 0.07 nM; Bmax = 14.7 fmol/mg protein). Binding was not affected by various spinal cord opioids or peptides. Autoradiographic studies indicated that binding sites were mainly located in the most external layers of dorsal horn where high densities of NPFF binding sites have previously been described. [125I]YLFQPQRFamide and [125I]DYLMeFQPQRFamide binding sites were both GTP-regulated. These findings indicate that DYLMeFQPQRFamide should be of value in studies on NPFF-mediated actions in vivo.