A major drawback to the use of OKT3, a mouse anti-CD3 monoclonal antibody (mAb), as an immunosuppressive agent is the associated cytokine release syndrome. We used a mouse model to elucidate the properties of anti-CD3 mAb responsible for these cytokine-related side effects. We have previously demonstrated that the hamster anti-CD3 mAb 145-2C11 induced strong cytokine release and morbidity in vivo, whereas two rat anti-CD3 mAb 17A2 and KT3 did not. In the current study, we show that the mitogenic capacity of soluble anti-CD3 mAb in vitro correlates with their induction of side effects in vivo. Mitogenesis in vitro and tumor necrosis factor-α (TNF-α) release in vivo induced by anti-CD3 mAb could be inhibited by the anti-FcγR mAb 2.4G2, indicating that FcγR binding of anti-CD3 mAb is responsible for their mitogenic properties and for their induction of side effects. Importantly, the two non-mitogenic rat anti-CD3 mAb were equally capable of suppressing skin allograft rejection as the mitogenic hamster anti-CD3 mAb, suggesting FcγR binding of anti-CD3 mAb is not essential for their immunosuppressive properties. This suggestion is reinforced by our demonstration that administration of 2.4G2 in vivo did not interfere with immunosuppression of skin allograft rejection by 145-2C11. These findings suggest that clinical use of non-mitogenic anti-CD3 mAb will result in effective immunosuppression without cytokine-related side effects.