SoLute Carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene (pept2) structure and determined mRNA tissue distribution. An EST cDNA (IMAGE) corresponding to zebrafish pept2 was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3238 bp functional clone. The complete ORF was 2160 bp and encoded a 719 amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevis oocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter (K_0.5 for glycyl-L-glutamine ~18 µM at -120 mV and pH 7.5). Zebrafish pept2 gene was 19435 kb, thus being the shortest vertebrate pept2 fully characterized so far. Also, zebrafish pept2 exhibited 23 exons and 22 introns, while human and rodent pept2 genes contain 22 exons and 21 introns only. Zebrafish pept2 mRNA was mainly detected in brain, kidney, gut and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homologue to the higher vertebrate inner ear, of the adult fish. Zebrafish pept2 will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development.