Characteristics, day-night changes, guanosine 5′-O-(3-thiotriphosphate) (GTPγS) modulation, and localization of melatonin binding sites in the brain of a marine teleost, European sea bass Dicentrarchus labrax, were studied by radioreceptor assay using 2-[¹²⁵I]iodomelatonin as a radioligand. The specific binding to the sea bass brain membranes was rapid, stable, saturable and reversible. The radio-ligand binds to a single class of receptor site with the affinity (Kd) of 9.3±0.6 pM and total binding capacity (Bmax) of 39.08±0.86 fmol/mg protein (mean±SEM, n=4) at mid-light under light-dark (LD) cycles of 12:12. Day-night changes were observed neither in the Kd nor in the Bmax under LD 12:12. Treatment with GTPγS significantly increased the Kd and decreased the Bmax both at mid-light and mid-dark. The binding sites were highly specific for 2-phenylmelatonin, 2-iodomelatonin, melatonin, and 6-chloromelatonin. Distribution of melatonin binding sites in the sea bass brain was uneven: The Bmax was determined to be highest in mesencephalic optic tectum-tegmentum and hypothalamus, intermediate in telencephalon, cerebellum-vestibulolateral lobe and medulla oblongata-spinal cord, and lowest in olfactory bulbs with the Kd in the low picomolar range. These results indicate that melatonin released from the pineal organ and/or retina plays neuromodulatory roles in the sea bass brain via G protein-coupled melatonin receptors.