There has been relatively little research conducted on pineal melatonin production in laboratory mice, in part, due to the lack of appropriate assays. We studied the pineal and plasma rhythm, response to light, adrenergic stimulation, and metabolism of melatonin in CBA mice. With the use of a sensitive and specific melatonin RIA, melatonin was detected in the pineal glands at all times of the day >21 fmol/gland in CBA mice but not in C57Bl mice. Both plasma and pineal melatonin levels peaked 2 h before dawn in a 12:12-h light-dark photoperiod (162 ± 31 pM and 1,804 ± 514 fmol/gland, respectively). A brief light pulse (200 lx/15 min), 2 h before lights on, suppressed both plasma and pineal melatonin to near basal levels within 30 min. Exposure to light pulses 4 h after lights off or 2 h before lights on resulted in delays and advances, respectively, in the early morning decline of plasma and pineal melatonin on the next cycle. Administration of the β-adrenergic agonist isoproterenol (20 mg/kg) 2 and 4 h after lights on in the morning resulted in a fivefold increase in plasma and pineal melatonin 2.5 to 3 h after the first injection. In the mouse, unlike the rat, melatonin was shown to be metabolized almost exclusively to 6-glucuronylmelatonin rather than 6-sulphatoxymelatonin. These studies have shown that the appropriate methodological tools are now available for studying melatonin rhythms in mice.