ZFOR1 is a putative opioid receptor from zebrafish brain which has 66% homology with the mammalian delta-opioid receptor. When expressed in HEK293 cells ZFOR1 bound the non-selective opioid antagonist [(3)H]diprenorphine with high affinity. However, the binding of this ligand was not readily displaced by opioids selective for mu, delta or kappa opioid receptors (affinities>1000 nM). Rather non-selective ligands showed good affinity, as did the non-peptide delta-ligand BW373U86 (Ki 69 nM), the delta-antagonist naltrindole (Ki 28 nM) and the peptide beta-endorphin (Ki 37 nM). Agonist binding to the receptor encoded by ZFOR1 receptor stimulated the binding of [(35)S]GTPgammaS confirming coupling to G proteins. Study of the receptor should contribute to understanding of the evolution of the opioid system.