The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian mu-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary, Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the mu-selective agonist [D-Ala(2), MePhe(4)-Gly-ol(5)] enkephalin (DAMGO) (K-d = 0.63 +/- 0.15 nM) and for the nonselective antagonist naloxone (K-d = 3.1 +/- 1.3 nM), The receptor had negligible affinity for U50488 and [D-Pen(2), D-Pen(5)]enkephalin (DP-DPE), which are kappa- and delta-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 mu M DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone, Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1), The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level, Furthermore, our data indicate that the mu-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates; and that the pharmacological and functional properties of this receptor have been conserved over a period of approximate to 400 million years implying that it fulfills an important physiological role.