Melatonin (MLT) treatment in vivo has been shown to have immunomodulatory and anti-immunosenescent effects in the mouse model. In the present report, the in vitro effect of MLT on mitogen-induced lymphocyte proliferation and cytokine expression was evaluated in a rat model. Splenic lymphocytes were isolated from young (6 months) and old (24 months) F344 rats and were incubated with MLT in the presence or absence of mitogens. The proliferative response to concanavalin A (ConA) or PMA plus ionomycin was measured in splenocytes or T cells isolated from young and old rats. In addition, the induction of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production was measured in MLT-treated and untreated lymphocytes isolated from young and old rats. The ConA-induced lymphocyte proliferation and IL-2 expression were significantly lower and induction of IFN-gamma production was significantly higher in splenocytes and purified T cells isolated from old rats compared to splenocytes and T cells isolated from young rats. Treatment of lymphocytes with MLT did not significantly alter ConA-induced lymphocyte proliferation or IL-2 or IFN-gamma expression in lymphocytes isolated from either young or old rats. On the basis of these data, we conclude that in vitro MLT treatment had no immunomodulatory effect on lymphocytes from rats.