To study the effects and mechanisms of melatonin (MT) on immune responses in mice of different months. METHODS: Thymocyte proliferation and IL-2 activity were assayed by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) and activated mouse splenocyte proliferation methods, respectively; cAMP and methionine-enkephalin (met-Enk) level was determined by competitive protein binding assay and radioimmunoassay, respectively. RESULTS: The function of lymphocytes, obtained from BALB/c mice aged 6 and 11 months were decreased, which was restored by melatonin at the dose of 5 mg/kg or 30 mg/kg. In vitro, proliferation of lymphocytes in 11-month-old mice was decreased and cAMP level was increased. Melatonin (0.1nmol/L or 1mmol/L) had negative regulation to this. Forskolin (10 mmol/L) enhanced the cAMP level of lymphocytes in 2-and 11-month-old mice (P<0.01), which was antagonized partially by melatonin and this effect of melatonin was also abolished by pertussis toxin (1mg/L) completely. Melatonin (1mmol/L and 0.1nmol/L) increased the content of met-Enk of lymphocytes in 2-and 11-month-old mice, respectively (P<0.01), which was blocked by nifedipine (1 mmol/L). CONCLUSION: Melatonin exerted an effect on immune responses in mice of different months, which might be mediated by G protein-AC-cAMP signal transduction pathway and regulation of met-Enk level.