It has been demonstrated that the development of stable mixed lymphocyte chimerism is associated with alloimmune tolerance induction in vascularized bone marrow transplant (VBMT) recipients. The underlying mechanisms of immune non-responsiveness in tolerant VBMT chimeras remains unclear. Our VBMT model involves the transplantation of a parental donor limb (Lewis rats) onto a hybrid (Lewis x Brown Norway) F1 recipient. Tolerogenic mechanisms and cellular immune regulation to self and host allodeterminants were investigated during the early post-transplant phase of tolerance induction. Flow cytometric analysis of sIg+-depleted experimental peripheral blood lymphocytes from tolerant VBMT recipients demonstrated low level stable mixed immune chimerism. Chimeric cells tested for responsiveness against self-LEW determinants showed activated proliferation and immune dysregulation 30 days post-transplantation. However, direct immunocytolytic activity against LEW determinants was not found. Tolerant chimeras also demonstrated elevated cellular proliferation and cytolytic responses against host-specific BN allodeterminants at 30 days. Consistent with these in vitro findings, limited clinical signs compatible with GVH reactivity were evident in vivo at this time. Following this initial period, the tolerant VBMT animals returned to normal clinical condition and remained otherwise healthy throughout the study. Consistent with these results, VBMT chimeras then showed declining proliferative responses from the elevated values seen at 30 days against self-LEW determinants. Proliferative and immunocytolytic responses also decreased against host-specific BN allodeterminants from peak levels at 30 days. In conclusion, these results provide evidence that the initial phases of tolerance induction in VBMT chimeras consist of self- and alloimmune regulation that follow an early period of immune dysregulation. Sequential phases of immune dysregulation and re-regulation elucidated in VBMT stable mixed chimeras within the first 100-day period may represent important mechanisms of tolerance induction.