To identify the characteristics of the oscillator located in the pineal organ, we examined the effects of temperature and light on melatonin secretion rhythm using pineal organs in cultures. At 20°C, the melatonin rhythm was obvious: low secretion during the daytime and high during the nighttime. When the temperature was lowered from 20 to 10°C, the melatonin rhythm disappeared. When the temperature was returned from 10 to 20°C, the rhythm quickly reappeared. The plasma melatonin level was measured in living lampreys kept at 7°C to establish the melatonin profile at low temperature in vivo: secretion was not significantly different between daytime and nighttime. Under continuous light conditions, the melatonin elevation normally seen during the subjective night became obscure after 72 h. When the LD cycle was shifted by 6 h (phase-advanced or phase-delayed), the melatonin rhythm shifted to remain in the same phase relation to the LD cycle. This re-synchronization took several LD cycles. The results indicate that, in cultures, the melatonin secretion rhythm in the pineal organ of the lamprey is both light- and temperature-sensitive, and that in vivo, the melatonin rhythm is not the critical factor maintaining the locomotor activity rhythm of the lamprey. The role of the pineal organ and melatonin in the circadian organization of the lamprey is discussed.