Neuropeptide FF (NPFF) receptors have been characterized in mouse olfactory bulb membranes by using [125I][1DMe]Y8Fa. The specific binding of this NPFF analogue was time and concentration dependent, reversible, saturable, and of high affinity (Kd = 0.022 nM, Bmax = 56.4 fmol/mg protein). In olfactory bulb membranes, NaCl increased the affinity of [125I][1DMe]Y8Fa by decreasing the dissociation rate constant (k-1). In contrast, the nonhydrolyzable analogue of GTP, Gpp[NH]p, decreased the maximal number of binding sites suggesting a coupling of NPFF receptors to a G-protein. In mouse olfactory bulb and spinal cord membranes, NPFF analogues stimulated adenylate cyclase activity in a time- and dose-dependent manner, whereas in the cerebellum, which does not possess NPFF receptors, low cAMP production was stimulated by NPFF. Our data are consistent with guanine nucleotide binding protein regulation of NPFF receptors positively coupled to adenylate cyclase.