The rhythm of melatonin in rat milk and the capacity of pups to synthesize and metabolize melatonin were studied. Melatonin was undetectable in milk in the light (<21 pM), but increased rapidly 2-4 h after dark to peak at 357 ± 66 pM at mid-dark. Oral or subcutaneous administration of melatonin to 5- and 10-day-old pups resulted in peak plasma melatonin levels 30 min after administration and rapid metabolism. Increases in pineal and plasma melatonin levels at night were detected at 5 and 6 days of age, respectively. Isoproterenol administration (2 µg/g body wt) at mid-light to day 10 pups increased plasma melatonin from 312 ± 40 pM to 1,298 ± 160 pM, whereas propranolol (2 µg/g body wt) suppressed nocturnal melatonin secretion from 1,270 ± 128 pM to 395 ± 66 pM. The rise of pineal and plasma melatonin in day 10 pups occurred 1 and 2 h after dark onset, respectively, preceding the onset in dams by 3 and 4 h, respectively. Propranolol administration to 2- and 5-day lactating dams inhibited plasma and milk melatonin at night but had no effect on their suckling pups. Transfer of melatonin via the milk is unlikely to provide an entraining signal for rat pups.