Background and Purpose The G protein-coupled receptor GPR173 (SREB3) regulates gonadotropin secretion and ovarian cyclicity. However, the identity of a ligand for this receptor remains uncertain. Recently, it was proposed that the cholecystokinin octapeptide CCK8 is a ligand for GPR173. Experimental Approach Here, comparison of responses to CCK8 in CHO-K1 cells transfected with human GPR173 or with with an empty vector indicate that responses to CCK8 are mediated by endogenous CCK receptors. Therefore, to further investigate CCK-type peptides as candidate ligands for GPR173-type receptors, we employed an evolutionary approach, informed by the ancient origin and co-evolution of neuropeptides and their cognate receptors. GPR173-type receptors were identified in two invertebrate species, the starfish Asterias rubens and the octopus Octopus vulgaris, and effects of CCK-type peptides from these species on CHO-K1 cells transfected with respective GPR173-type receptors or empty vectors were investigated Key Results Starfish and octopus CCK-type peptides had no effect on CHO-K1 cells transfected with empty vectors or with respective GPR173-type receptors. However, positive control experiments demonstrated that starfish and octopus CCK-type peptides triggered responses in CHO-K1 cells transfected with respective orthologs of mammalian CCKA/CCKB-type receptors. Conclusion and Implications Our findings indicate that CCK-type neuropeptides do not act as ligands for GPR173 in humans or GPR173-type receptors in other taxa. Furthermore, this study underscores the importance of an evolutionary approach in assessing candidate ligands for orphan receptors, informed by phylogenetic analysis of the distribution of ligands and receptors, thereby avoiding challenges associated with endogenous receptor expression in mammalian cell lines