Anti-CD3 mAbs are potent inhibitors of T cell function; however, administration of mitogenic mAb can cause significant morbidity secondary to T cell activation and cytokine release. Nonmitogenic anti-CD3 mAb is immunosuppressive in mice without inducing detectable morbidity, and may thus be preferable for in vivo T cell immunosuppression. The precise mechanisms of action of these two forms of mAb have not been fully defined. To further characterize and compare the in vivo functional effects of mitogenic and nonmitogenic anti-CD3 mAbs, mice were treated with the intact (mitogenic) form of the anti-murine CD3 mAb, 2C11, or with F(ab')2 fragments (nonmitogenic). Effects on T cell phenotypes and the secretion of Th1-derived cytokines were compared. Nonmitogenic mAb induced a prolonged downregulation of secretion of interleukin (IL)-2 and interferon (IFN)-gamma from CD4+ T cells, and of IL-2 secretion from CD8+ T cells, and preferential depletion of CD4+ T cells. In marked contrast, mitogenic mAb induced a prolonged upregulation of IL-2 and IFN-gamma secretion from both CD4+ and CD8+ cells, and preferential depletion of CD8+ T cells. Both forms of mAb induced a shift in the T cell populations from a naive to a memory phenotype; however, this shift was not responsible for the observed changes in cytokine secretion. These results demonstrate that mitogenic and nonmitogenic forms of 2C11, while binding to the identical epitope, differentially affect T cell functions, and have implications for the use of anti-CD3 mAbs in the clinical setting.