AbstractGonadotropin-releasing hormone (GnRH) is a key regulator of reproductive function in vertebrates. GnRH is related to the corazonin (CRZ) neuropeptide which influences metabolism and stress responses in insects. Recent evidence suggests that GnRH and CRZ are paralogous and arose by a gene duplication in a common ancestor of bilaterians. Here, we report the identification and complete characterization of the GnRH and CRZ signaling systems in the amphioxusBranchiostoma floridae. We have identified a novel GnRH peptide (YSYSYGFAP-NH2) that specifically activates two GnRH receptors and a CRZ peptide (FTYTHTW-NH2) that activates three CRZ receptors inB. floridae. The latter appear to be promiscuous, as two CRZ receptors can also be activated by GnRH in the physiological range. Hence, there is a potential for cross-talk between these closely related signaling systems. Discovery of both the GnRH and CRZ signaling systems in one of the closest living relatives of vertebrates provides a framework to discover their roles at the transition from invertebrates to vertebrates.