The melanocortin 5 receptor (MC5R) is activated by melanocyte-stimulating hormones (MSHs) and has a widespread tissue distribution, while its detailed central expression pattern and brain functions are fairly unknown. We report cloning, pharmacological characterization, tissue distribution and detailed brain mapping of melanocortin 5 receptor in goldfish (gMC5R). The goldfish orthologue protein is 69% identical to human MC5R and is conserved in important functional domains. The gMC5R showed similar potency to alpha-, beta- and gamma-MSH peptides in radioligand binding as the mammalian orthologues, while MTII and HS024 were both agonists at this receptor. The gMC5R-mRNA was found in the peripheral tissues including kidney, spleen, skin and retina, with low expression levels in the intestine, fat, muscle, gill, pituitary and ovary. In situ hybridization studies demonstrated that gMC5R transcripts are widely distributed in the goldfish brain. The gMC5R expression was found in ventral telencephalon, pre-optic area, dorsal and ventral thalamus, infundibular hypothalamus, posterior tuberculum, tectum and tegmentum mesencephali, reticular formation, vagal and facial lobes and spinal cord. The cloning and characterization of this receptor provides an important tool to elucidate its participation in neuroendocrine and behavioural control.