In fish, as well as in birds and mammals, the pineal hormone melatonin acts as a neuroendocrine transducer of photoperiod and as such has a profound influence over the timing of seasonal behavior, especially reproduction. With the aim of better understanding how melatonin may exert its influence in the fish, we have studied the distribution and characteristics of the melatonin binding sites in the brain and pituitary of the goldfish (Carassius auratus) using the ligand [2-(125)I]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of the ligand was defined as that displaced by 1 muM melatonin. High-affinity melatonin binding sites were found to have a widespread distribution within the brain of the goldfish and measurements of the relative optical densities on X-ray films using image analysis showed that the highest concentrations were present in the visual system (optic tract, pretectal area, optic tectum), the molecular layer of the cerebellum, and the hypothalamus. No specific binding could be detected in either the pituitary gland or the olfactory bulbs. Displacement of binding by increasing concentrations of melatonin measured by comparative optical density readings on serial sections revealed an ED(50) for melatonin of 10(-9)M. No displacement of binding by increasing concentrations of serotonin up to a concentration of 10(-6) M could be detected. Binding was shown to be time dependent with maximal binding occurring after 1 h at room temperature (22 degrees C). Addition of 1 muM melatonin after 2 h resulted in a time-dependent dissociation of ligand binding. Saturation studies carried out on crude membrane homogenates of whole brains gave rise to a straight line Scatchard plot with a K(D) of 69 pM and a B(max) of 14 fmol/mg protein. The widespread distribution of high-affinity melatonin binding sites in the brain of the goldfish suggests that melatonin may influence circadian and seasonal behavior in fish via numerous sites involving sensory and neuroendocrine systems but does not have a direct action on the pituitary.