The factors associated with the development of humoral sensitization were studied prospectively in 30 previously transplanted patients immediately after graft rejection. Lymphocyte antibodies were measured both by conventional cytotoxicity in 30 panel cells and by flow cytometry in up to 10 target cells. Although lymphocyte antibodies induced by graft rejection alone were detected in 12 of 26 patients (46%), lymphocytotoxic antibodies were present in only 2 of 27 patients. Of the 25 patients without lymphocytotoxic antibodies, 13 developed them later. In all cases panel antibody reactivity developed after the patients received blood transfusions. No other factor was associated with the development of lymphocytotoxic antibodies, including transplant nephrectomy. There were 12 patients who remained negative for lymphocytotoxic antibodies even though 5 of them were transfused. The powerful role of blood transfusions in the generation of broad sensitization was further documented in 5 patients who received blood units completely depleted of leukocytes by cottonwool filtration and red cell washing. Four of these patients showed significant increases in the level of lymphocytotoxic antibodies, even when stored blood units were used. One additional patient became broadly sensitized by the transfusion of frozen blood. These results show (A) that broad sensitization may not develop if patients are not transfused after graft rejection; (B) that blood transfusions lead to broad sensitization in most (76%) pretransplanted patients; and (C) that transfusion of leukocyte-free blood may delay, but not avoid, the development of broad sensitization.