The KISS1 gene encodes the kisspeptin neuropeptide, which activates the KISS1 receptor (KISS1R; G protein-coupled receptor 54; GPR54) and participates in neuroendocrine regulation of GnRH secretion. To study the physiological function(s) and evolutionary conservation of KISS1, we cloned opossum, Xenopus, and zebrafish kiss1 cDNAs. Processing zebrafish, Xenopus, or opossum KISS proteins would liberate a carboxy-terminal amidated peptide with 52, 54, or 53 amino acid residues, respectively. Phylogenetic analysis of all known vertebrate KISS1 peptides showed clear clustering of the sequences according to canonical vertebrate classes. The zebrafish kiss1 gene consists of two exons and one intron. Real-time PCR analysis of two kiss1R cloned from zebrafish brain found expression of kiss1, kiss1ra, and kiss1rb, with kiss1ra—more similar to other piscine Kiss1 receptors—highly expressed in the gonads and kiss1rb in other nonbrain tissues. In females kiss1 mRNA levels gradually increased during the first few weeks of life to peak in fish with ovaries containing mature oocytes, while in males kiss1 mRNA levels peaked after 6 wk postfertilization when the testes exhibited initial stages of spermatogenesis and decreased after puberty. Zebrafish kiss1ra and kiss1rb were expressed differentially with similar patterns in both genders. These results indicate that the Kiss1/Kiss1r system may participate in puberty initiation in fish as well. Like human KISS1R, Kiss1ra transduces its activity via the PKC pathway, whereas Kiss1rb does so via both PKC and PKA pathways. The human KISS1R was highly activated by both huKISS10amide and zfKISS10amide, whereas both zebrafish Kiss1 receptor types were less sensitive to amidation.