By using an optimized [35S]GTPγS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF1 and NPFF2 receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF1 and NPFF2 receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF2 receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF1 receptors, NPFF2 receptors exhibited an extraordinary activity since all compounds tested displayed EC50 values of GTPγS binding lower than their KI values. Comparisons of functional values between NPFF1 and NPFF2 receptors revealed unexpected potent selective NPFF2 agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Gαi3 and Gαs are the main transducers of NPFF1 receptors while NPFF2 are probably coupled with Gαi2, Gαi3, Gαo and Gαs proteins. Our data indicate that NPPF1 and NPFF2 receptors are differently coupled to G proteins in CHO cells.