GPR7 and GPR8 are two structurally related orphan G protein-coupled receptors, presenting high similarities with opioid and somatostatin receptors. Two peptides, L8 and L8C, derived from a larger precursor, were recently described as natural ligands for GPR8 (Mori, M., Shimomura, Y., Harada, M., Kurihara, M., Kitada, C., Asami, T., Matsumoto, Y., Adachi, Y., Watanabe, T., Sugo, T., and Abe, M. (December, 27, 2001) World Patent Cooperation Treaty, Patent Application WO 01/98494A1). L8 is a 23-amino acid peptide, whereas L8C is the same peptide with a C terminus extension of 7 amino acids, running through a dibasic motif of proteolytic processing. Using as a query the amino acid sequence of the L8 peptide, we have identified in DNA databases a human gene predicted to encode related peptides and its mouse ortholog. By analogy with L8 and L8C, two peptides, named L7 and L7C could result from the processing of a 125-amino acid human precursor through the alternative usage of a dibasic amino acid motif. The activity of these four peptides was investigated on GPR7 and GPR8. In binding assays, L7, L7C, L8, and L8C were found to bind with low nanomolar affinities to the GPR7 and GPR8 receptors expressed in Chinese hamster ovary (CHO)-K1 cells. They inhibited forskolin-stimulated cAMP accumulation through a pertussis toxin-sensitive mechanism. The tissue distribution of prepro-L7 (ppL7) and prepro-L8 (ppL8) was investigated by reverse transcription-PCR. Abundant ppL7 transcripts were found throughout the brain as well as in spinal cord, spleen, testis, and placenta; ppL8 transcripts displayed a more restricted distribution in brain, with high levels in substantia nigra, but were more abundant in peripheral tissues. The ppL7 and ppL8 genes therefore encode the precursors of a class of peptide ligands, active on two receptor subtypes, GPR7 and GPR8. The distinct tissue distribution of the receptor and peptide precursors suggest that each ligand and receptor has partially overlapping but also specific roles in this signaling system.