Relaxin-3 (R3) is the most recently identified member of the insulin superfamily, which is composed of peptides with diverse sequences held together by characteristic disulfide links connecting A and B peptide chains. R3 has nearly exclusive expression in the brainstem. It was demonstrated to be an additional ligand for the relaxin receptor LGR7, which is a class-C hormone receptor type G-protein coupled receptor (GPCR). We recently identified R3 as a ligand for two orphan G-protein coupled receptors, GPCR135 (aka SALPR) and GPCR142 (aka GPR100), which are class-A GPCRs and typical neuropeptide receptors. The predominant brain expression for both R3 and GPCR135, coupled with their high affinity interaction, strongly suggests that R3 is the endogenous ligand for GPCR135. Both R3 and GPCR135 from different species are highly conserved from genetic sequences to in vitro pharmacology. In contrast, GPCR142 is a pseudogene in rats, and the mouse gene is less conserved with human GPCR142, suggesting that GPCR142 may have a diminished role as a receptor for R3 in rodents. Further studies of GPCR142 in monkeys, cows, and pigs demonstrate that GPCR142 in those species shares high homology to the human GPCR142, and that it behaves similarly to the human receptor in vitro. This suggests that GPCR142 has conserved functions in these non-rodent species, including humans. In addition, the tissue expression pattern of GPCR142, primarily in peripheral tissue, is drastically different from R3, suggesting that GPCR142 may have an endogenous ligand other than R3. Sequence analysis among insulin/relaxin family members shows that insulin-like peptide 5 (INSL5) is the closest member to R3. Pharmacological characterization shows that INSL5 is a specific agonist for GPCR142, but not for GPCR135. Specifically, INSL5 binds to and activates GPCR142 at high affinity. Although INSL5 binds to GPCR135 at low affinity, it does not activate GPCR135. INSL5 mRNA is primarily expressed in the periphery, and its expression pattern overlaps with that of GPCR142, consistent with INSL5 being the endogenous ligand for GPCR142. Endogenous ligands and receptors tend to co-evolve. Consequently, INSL5, like GPCR142, is a pseudogene in rats, which further implies that INSL5/GPCR142 is an endogenous ligand/receptor pair. R3 can activate GPCR135, GPCR142, and LGR7. Therefore, in vivo administration of R3 could potentially activate all three receptors, which complicates the functional studies of GPCR135. By substituting the A chain of R3 with the A chain of INSL5, we devised a chimeric peptide (R3/I5), which is about 1000-fold more selective for GPCR135 and GPCR142, than for LGR7. C-terminal truncation of this chimeric peptide resulted in a potent antagonist [R3(BDelta23-27)R/I5] for GPCR135 and GPCR142, with no affinity for LGR7. The selective agonist and antagonist pair is particularly helpful for in vivo studies of GPCR135 in rats lacking GPCR142. R3 is highly expressed in the nucleus incertus, a region of the brain stem, which has been known to send afferent connections to different brain regions. [125 I]R3/I5 is a radioligand that has an improved signal/noise ratio compared to [125 ]R3. Autoradiographic distribution of GPCR135 binding sites using [125 I]R3/I5 in rat brain shows that GPCR135 receptor is prominent in many regions, including olfactory bulb, amygdala, thalamus, somatosensory cortex, and superior colliculus, which have been reported to have connections to the nucleus incertus. Different brain regions serve different functions. The expression pattern of R3 and GPCR135 in the brain suggests multiple functions of R3 and GPCR135. The high level expression of R3 in the brainstem co-localizes with the expression of corticotrophin releasing factor receptor 1 (CRF1), suggesting a potential role of R3/GPCR135 in stress response. Water-restraint stress-induced R3 mRNA expression in the brain stem seems to support this hypothesis. In addition, recent studies have shown that acute and chronic intracerebroventricular (i.c.v.) administration of R3 induces feeding in rats. More specifically, i.c.v. injection of R3/I5 (GPCR135 selective agonist) stimulates feeding in rats, an effect that can be blocked by the GPCR135-selective antagonist R3(BDelta23-27)/I5, thus confirming the involvement of R3 and GPCR135 in feeding. The availability of those pharmacological tools should greatly facilitate future studies of the physiology of GPCR135 and GPCR142.