Melatonin release was measured from zebrafish pineal organs and retinas maintained in flow-through culture. Pineal organs released melatonin in a strong circadian rhythm through 5 days in constant darkness, and the phase of this rhythm was reset by in vitro exposure to phase-shifted light cycles. In contrast, the retinal melatonin rhythm rapidly damped out in constant darkness, even in the presence of (phase-shifted) light cycles. The zebrafish pineal should be useful for in vitro studies of vertebrate circadian clock mechanisms.