A circadian pacemaker, thought to be within the suprachiasmatic nucleus (SCN) of the hypothalamus, begins to function before birth in rodents. Prenatal entrainment of the pacemaker appears to be mediated by signals regulated by the maternal SCN; ablation of the mother's SCN during gestation disrupts the normal phase of the pups' rhythms. The present paper presents an experimental approach for identifying candidate entraining signals and for testing when they are effective during development. The candidate signal examined in these experiments was the pineal gland hormone, melatonin. Female golden hamsters (Mesocricetus auratus) received SCN lesions on day 7 of gestation. During the last week of gestation, they were given two daily subcutaneous injections of oil 12 h apart. One of the injections each day contained melatonin (10, 50, or 100 micrograms). The phases of the pups' activity rhythms were measured at weaning and were found to be related to the timing of the daily injection that contained melatonin, demonstrating that the melatonin directly or indirectly set the phase of the pups' rhythms. Injections given over 4 days of gestation were found to be as effective as injections given over 7 days. Although a physiological role for melatonin as an entraining signal has not been demonstrated, the results show that exogenous, prenatal treatment can predictably set the phase of the offsprings' circadian rhythms.