Cloned Th1 cells, but not Th2 cells, specific for malaria antigen, produce nitric oxide (NO) when activated with specific antigen or Con A. Furthermore, NO inhibits proliferation of, and production of IL-2 and IFN-gamma by, Th1 but not Th2 cells. Here, it is demonstrated that the inhibition of Th1 cell proliferation by the NO donor S-nitroso-N-acetyl penicillamine (SNAP) can be reversed by the addition of rIL-2 but not of rIFN-gamma, suggesting that the inhibition of Th1 cells by NO may be preventing the production of IL-2. Dose-response studies showed that Th1 cells produce optimal levels of IL-2 and a proliferative response, and no detectable NO, when stimulated with relatively low concentrations of antigen or mitogen in vitro. As the antigen/mitogen increased, however, high levels of NO were produced, accompanied by a concomitant reduction in IL-2 secretion and T cell proliferation. The proliferation of, and IL-2-IFN-gamma production by, naive CD4+ T cells from normal spleens activated with Con A in vitro can be similarly inhibited by SNAP. These results suggest that NO may serve as a self-regulatory molecule preventing the over-expansion of Th1 cells. Unrestricted Th1 cell activity has been implicated in a range of immunopathologies and autoimmune diseases. The proposed mechanism for down-regulation of Th1 cell function may also account for the suppression of lymphocyte proliferation observed in malaria infections.