A new full-length cDNA (ZFOR4) that encodes an opioid receptor has been isolated from the teleost zebrafish. The encoded polypeptide is 375 amino acids long and shows high sequence similarity to other δ-opioid receptors, including ZFOR1, the other δ-opioid receptor from zebrafish previously characterized by us. In situ hybridization studies have revealed that ZFOR4 mRNA is highly expressed in particular brain areas that coincide with the expression of the δ-opioid receptor in other species. Pharmacological analysis of ZFOR4 shows specific and saturable binding with [3H] diprenorphine, displaying one binding site with KD=3-42±0.38 nM and a receptor density of 6231 ± 335 fmol/mg protein. Competition-binding experiments were performed using [3H]diprenorphine and several unlabelled ligands (peptidic and non-peptidic). The order of affinity obtained is Met-enkephalin > Naloxone > Leu-enkephalin > Dynorphin A ≪ BW373U86 > Morphinex ⋙ [D-Pen2 D-Pen5]-Enkephalin, U69,593. [35 S]GTPγS stimulation studies show that the endogenous ligands Met- and Leu-enkephalin and the non-peptidic δ agonist BW373U86 were able to fully activate ZFOR4. Our results prove the existence of two functional duplicate genes of the δ-opioid receptor in the teleost zebrafish.