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, newly developed 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 post-transplant 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 during daily serologic and laboratory monitoring. 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. All three cases with hemolysis involved female donors, compared with only two of seven cases 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. Immune hemolysis can adversely impact the otherwise improved safety profile of nonmyeloablative conditioning regimens. Serologic monitoring alone was not sufficient to determine cases with from those without hemolysis. Twice daily blood counts during the period at risk (day 6 to 11 post-transplant), careful monitoring of red cell serologic studies (DAT, IAT), prompt donor-compatible red cell transfusions, and improved awareness can avert serious complications due to minor ABO incompatibility and should be practiced in all such cases.