Although pinealectomy has little effect on the generation of circadian rhythmicity by mammals, daily injections of the pineal hormone melatonin entrain free-running rats [30]. The present study was designed to determine if known components of mammalian circadian organization were necessary for melatonin entrainment. Rats received either lesions to the suprachiasmatic nuclei (SCN), sham-lesions or neurotoxic lesions to brain catecholamines or serotonin. They were then allowed to free-run in constant dim red light (DD) before each received daily injections of either 1 mg/kg melatonin or ethanol:saline vehicle for 90 days. They were allowed to free-run for 30 days afterwards. Rats which received sham-lesions or neurotoxic lesions entrained to melatonin injections but not to vehicle. Rats which received complete SCN lesions were unaffected by melatonin or vehicle. These data suggest that the behavioral effects of melatonin, like those on reproduction in seasonally breeding mammals, depend upon an intact circadian system and the SCN.