Rabbitfishes are known to spawn synchronously around the species-specific lunar phase. It is considered that they perceive and utilize cues from the moon in order to be synchronized gonadal development and spawning with the lunar cycle. Using the golden rabbitfish, Siganus guttatus, which spawns synchronously around the first quarter moon during the reproductive season, we measured the fluctuation of melatonin levels and examined the response of the fish to moonlight intensity. Daily fluctuation of melatonin concentration in the blood of golden rabbitfish showed low levels during daytime and high levels during night-time, suggesting that melatonin functions in the perception and utilization of photoperiod. Plasma melatonin concentration at the new moon was higher than that at the full moon. When the fish were exposed to moonlight at midnight of the both moon phases, the melatonin concentrations decreased to the control levels. These results show that the fish possibly perceive moonlight intensity and plasma melatonin fluctuates according to ‘lightness’ at a point of night. At the first spawning period (experiment was started one month before the spawning), the fish reared under natural conditions spawned at the expected spawning dates, whereas the fish reared under the constant darkness and lightness of night did not spawn. At the second spawning period (experiment was started 2 weeks before the spawning), the fish reared under the conditions of natural and constant darkness of night spawned but not that of constant lightness of night. It is possible that night conditions are related to synchronous gonadal development and spawning in the golden rabbitfish. J. Exp. Zool. 301A:844–851, 2004. © 2004 Wiley-Liss, Inc.