During murine Trypanosoma brucei infection, macrophages contribute significantly to the inhibition of T cell responses. Although nitric oxide (NO) was shown to play a central role in macrophage-mediated splenic suppression, macrophage-mediated lymph node suppression occurred in an interferon-gamma (IFN-gamma)-dependent manner. In this study, using NO inhibitor NG-monomethyl-L-arginine and anti-IFN-gamma antibodies, the relative contribution of NO and IFN-gamma to the active inhibition of ex vivo concanavalin A-induced T cell proliferation taking place in the spleen and the lymph nodes of T. brucei-infected mice was investigated. NO contributes to the suppressive activity of spleen and lymph node cells only during early-stage infection. The existence of NO-independent suppressive pathway was further evidenced in IFN-gamma(-/-)-infected mice. Spleen cells from such animals do not produce NO but exert significant suppressive activity during the whole course of infection. In contrast in the lymph nodes, no suppressive activity is recorded at any moment of infection. Moreover, addition of exogenous IFN-gamma to cultures containing lymph node cells from IFN-gamma(-/-)-infected mice does not impair proliferation despite NO production in such cultures. Thus during late-stage infection, an IFN-gamma-independent suppressive mechanism is elicited in the spleen, whereas in the lymph nodes, IFN-gamma is required yet not sufficient to inhibit T cell proliferation.