The 24-hour rhythm of retinal and hypothalamic melatonin immunofluorescence was determined in male albino rats and compared to the 24-hour rhythm of serum melatonin determined by radioimmunoassay. Under a 12-hour (0:00–12:00):12-hour dark cycle, the 24-hour rhythm of melatonin immunocytochemical fluorescence in the retina was bio-modal (crests at 1:00 and 10:00–13:00). In serum, melatonin has a single crest late in the dark period (18:00). In the hypothalamus melatonin immunofluorescence showed increments corresponding to the crests in retina (13:00) and serum (18:00). In a 2-hour light: 22-hour dark cycle, the retinal rhythm was suppressed, the serum rhythm unchanged, and the hypothalamic rhythm had a single crest corresponding to the crest of the serum melatonin rhythm. These data indicate that the 24-hour hypothalamic rhythm of melatonin immunofluorescence may be secondary to the serum and retinal 24-hour rhythms of melatonin.