The spectrum of pediatric bone marrow transplantation has changed in recent years. Mismatched and unrelated donor transplants are common, demanding an increased vigilance to detect incipient graft failure, secondary lymphoma as well as relapse and other problems, which now are potentially treatable. To diagnose these complications it may be important to know whether blood and marrow cells are of recipient or donor origin. To evaluate the role of mixed donor-recipient chimerism in relation to clinical problems we adapted a polymerase chain-reaction technique, using fluorescent primers analyzing DNA fragment length polymorphisms, to follow prospectively 17 bone marrow grafted children. To increase the precision of chimerism analysis, immunomagnetically isolated leukocyte populations were assayed in selected cases. Five patients encountered clinical problems related to chimerism. One infant with adenosine deaminase deficiency failed to engraft stem cells, yet succumbed to graft-versus-host disease, mediated by mature donor T-cells. Three children developed significant mixed chimerism. One of these three patients died in relapse of leukemia, while the two other patients who had received T-cell depleted grafts had persistent recipient T-cells, in spite of engraftment. After 5 months, these were displaced by donor T-cells in one of the patients. In the fifth patient, also after T-cell depleted BMT, a fatal donor cell lymphoma occurred. Twelve children had stable full chimerism or in one case a low grade mixed chimerism and remain disease-free throughout follow up (median 9 months). In conclusion, the analysis of chimerism, particularly of separated leukocyte populations, offers an almost indispensable insight and a basis for therapeutic decisions in complicated situations such as grafting involving unrelated or mismatched donors, graft manipulation, adoptive immunotherapy and in immunodeficiency patients.