Neuropeptide FF (NPFF) and prolactin-releasing peptide (PrRP) are two members of the RFamide peptide family. In this study we investigated whether these RFamide peptides, which have common structural features in their C-terminal RFamide motif and share several physiologically important functions, could exert their effects through the same set of receptors. The affinity and functional activity of several related RFamide peptides were determined at the human neuropeptide FF receptor subtype 2 (hNPFF2) and the human prolactin-releasing peptide (hPrRP) receptors. The full-length human prolactin releasing peptide 31 (hPrRP31) had significantly higher efficacy compared with NPFF and its stable analog, (1DMe)Y8Fa, at the hNPFF2 receptor. In contrast, NPFF and (1DMe)Y8Fa were not efficacious at the hPrRP receptor. Our study indicated a generally relatively low level of discrimination for RFamide peptides at the NPFF receptor, whereas the hPrRP receptor clearly preferred PrRP or very closely related peptides. The seemingly promiscuous binding of the RFamide peptides to the NPFF receptor was further confirmed by receptor autoradiography. PrRP may thus signal through the NPFF receptors in vivo.