The source of donor alloantigen required to maintain tolerance in vivo was evaluated in anti-Cd4 monoclonal antibody (mAb) treated mice. Treatment with a depleting anti-Cd4 mAb at the time of transplantation (day-1, 0) induces tolerance to C57BL/10 (H2 b) vascularized cardiac allografts in C3H. He (H2 k) mice (1). The presence of the vascularized allograft was found to be essential for the induction of tolerance in this experimental model; it is the only source of donor alloantigen during the induction phase of unresponsiveness (0-50 days). In the maintenance phase (> 50 days) donor alloantigen is potentially available from two sources, the organ graft itself or donor cells that have migrated out of the graft and are resident in the periphery (donor microchimerism). We show that the vascularized cardiac allograft is essential for the maintenance of tolerance to donor alloantigen in vivo. When the primary heart graft remained in situ, tolerance to donor alloantigens, as assessed by the survival of a second heart graft, was maintained indefinitely (> 250 days)(MST of second C57 heart grafts> 100 days). However, when the primary heart graft was removed 50 days after transplantation, a time point when tolerance to donor alloantigens was demonstrable in vivo, tolerance was lost 200 days later (MST of second C57 heart grafts 31 days). No evidence of donor microchimerism in the recipient was obtained using allele specific polymerase chain reaction (pcr) analysis for donor class I antigen. Persistence of donor alloantigen in the form of the vascularized organ graft is therefore required for both the induction and maintenance of tolerance to alloantigen in vivo in this experimental model.