Administration of monoclonal antibodies (mAb) to CD3 elicits an immune response to the mAb and an acute toxic syndrome that has been attributed to the release of cytokines from activated T cells. To clarify the cellular basis for these effects, we used anti-lymphocyte mAb to deplete selected T-cell subsets from BALB/c mice prior to administration of anti-CD3. In our first series of experiments, anti-CD4 repeatedly blocked the immune response to anti-CD3, but did not prevent severe toxicity. This observation suggested that other T-cell subsets might contribute to anti-CD3 induced toxicity. Therefore, we treated mice with mAb to CD8 as well as mAb to CD4 prior to administration of anti-CD3. Despite depletion of > 95% of CD8+ and CD4+ T cells, toxicity was not suppressed. This finding cast doubt on the belief that toxicity is due to activation of either CD4+ or CD8+ T cells by anti-CD3. Therefore, we assessed the role of thymocytes (which are not deleted by the mAb) and gamma delta + T cells. Thymectomy did not prevent toxicity in CD4/CD8-depleted mice, demonstrating that thymocytes are not responsible for toxicity. Anti-alpha beta TCR mAb produced a toxic reaction similar to anti-CD3 whereas anti-gamma delta TCR mAb did not, suggesting that gamma delta+ T cells are not the source of toxic cytokines. In addition, we proved that anti-CD3-induced toxicity was not due to direct effects on macrophages or to other nonspecific factors associated with the hamster mAb. These findings imply that a few residual mature T cells in mice treated with mAb to CD4 and CD8 are sufficient for the full expression of the anti-CD3-induced toxic syndrome. To confirm that both CD4+ and CD8+ T cells can mediate toxicity, we showed that:(i) SCID mice, which normally do not develop anti-CD3-induced toxicity, can be rendered susceptible by reconstitution with purified CD4+ T cells; and (ii) CD4-knockout mice that lack CD4+ T cells but have normal CD8+ T cells are susceptible to anti-CD3-induced toxicity. These findings establish that both CD4+ and CD8+ cells contribute to the toxic effects of anti-CD3, and that relatively few cells are required to mediate the full effect.