The combination of a depleting anti-Cd4 3 monoclonal antibody (mAb) and a single donor-specific transfusion before transplantation has been shown to induce operational transplantation tolerance in the majority of cardiac allograft recipients in a mouse model. To examine a protocol which might be more clinically relevant, we have modified this tolerance-inducing protocol by substituting the depleting with a nondepleting anti-Cd4 mAb. We show that this form of pretreatment can also induce immunologic unresponsiveness in most recipients (C3H/He, H2 k), provided a critical period of time, in this case 28 days, is allowed between pretreatment and transplantation of a fully mismatched heart graft (H2 b). When only 1 or 2 weeks were allowed between pretreatment and transplantation, only slight graft prolongation was obtained when compared with recipients receiving anti-Cd4 mAb alone, at these time points. Maintenance of tolerance in this model was due, at least in part, to active mechanisms as immunologic unresponsiveness to donor antigens could be transferred to naive syngeneic mice by splenocytes from recipients bearing long-term functioning grafts. These findings suggest that a population of regulatory cells develop after pretreatment with nondepleting anti-Cd4 mAb and donor-specific transfusion, and that it takes at least 1 month for these cells to expand and effectively drive the recipient's immune system toward immunologic unresponsiveness.