Background. Donor-directed antibodies (DDA) have been shown to result in poor graft survival. This Study was designed to analyze antibody appearance and patient and graft characteristics related to antibody formation in patients who lost their graft at different time points after transplantation. Methods. Pre- and posttransplant sera of 56 DDA-negative first transplant patients were screened for human leukocyte antigen (HLA) class I and II DDA by the Luminex single antigen assay (LSA). All patients were treated with calcineurine inhibitor-based immunosuppression. Results. Three of 56 patients proved DDA positive by LSA before transplantation. Eighty-one percent of the remaining 53 patients became DDA class I or II positive or both; 16% before and 84% after transplantectomy. Class I antibodies were produced in 84% and class 11 in 77% of the recipients. Based on time of transplantectomy, three groups were created as follows: less than or equal to I month, 1 to 6 months, and more than 6 months. The groups proved to be significantly different for HLA class II mismatch and acute rejection. All recipients in group I to 6 months proved to be DDA positive. Logistic regression analysis showed that DDA positivity for class I was related to higher donor age and donor type (nonheart beating), class II to higher donor age and class II mismatch. Conclusions. Donor-directed HLA antibodies after transplantation were demonstrated in 81% of first transplant recipients, all of whom were DDA negative by LSA before transplantation. The majority of the antibodies was found after transplantectomy. These findings may have to be taken into consideration in the allocation of organs of marginal donors such as older or nonheart beating kidneys.