Massive immune hemolysis due to minor ABO incompatibility is an underappreciated, potentially fatal complication of allogeneic hematopoietic transplantation. The increased lymphoid content and rapid engraftment seen with peripheral blood stem cell (PBSC) transplants may increase the frequency and severity of this event. In addition, nonmyeloablative conditioning regimens favor rapid and vigorous donor-type immune reconstitution, relying on donor lymphocytes to mediate both an anti-tumor effect and durable myeloid engraftment. To further the graft versus tumor effect, antiproliferative agents such as methotrexate are frequently omitted from posttransplant anti-GVHD regimens. We observed abrupt, catastrophic hemolysis in the first NIH patient to receive a nonmyeloablative PBSC transplant involving minor ABO incompatibility. We established a protocol for close clinical and laboratory monitoring of the next nine consecutive minor ABO-incompatible, nonmyeloablative PBSC transplants performed on NHLBI and NCI services. Cyclosporine alone was employed to prevent GVHD in all nine cases. Two additional cases of massive immune hemolysis were detected. Hemolysis began 7 to 11 days following stem cell infusion. Both cases responded rapidly to vigorous hydration and prompt donor-compatible red cell transfusions, without adverse clinical consequences. All patients with hemolysis demonstrated a positive direct antiglobulin test (DAT), with eluate reactivity against the relevant recipient blood group (anti-A in two cases, anti-B in one). However, neither the intensity of the DAT nor the donor isohemagglutinin titer distinguished cases with from those without hemolysis. These results demonstrate that isohemagglutinins produced by donor passenger B lymphocytes in minor ABO incompatible, PBSC transplants utilizing cyclosporine alone for GVHD prophylaxis can mediate massive immune hemolysis in a considerable proportion of subjects at risk. In view of this high risk, anti-GVHD regimens in NHLBI protocols were changed to include mycophenolate mofetil (MMF), an antiproliferative agent. None of the next 10 consecutive minor ABO incompatible nonmyeloablative stem cell transplants was accompanied by significant immune hemolysis, although serologic abnormalities were seen. GVHD regimens continue to be modified to maximize graft anti-tumor immune effects while minimizing other immune complications of transplant, and MMF doses are being reduced in an effort to increase complete remission rates posttransplant. We continue to monitor daily blood counts and red cell serologic studies (DAT, IAT) during the period at risk (day 6 to 11 posttransplant) and to promptly administer donor-compatible red cell transfusions in these cases. Improved awareness can avert serious complications due to minor ABO incompatibility following stem cell transplant and should be practiced in all such cases.