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ABO-incompatible kidney transplantations require the anti-ABO antibody titer to be reduced to below 1:16 before transplantation. To achieve this, preoperative plasma exchange or double-filtration plasmapheresis (DFPP) is usually performed. We report a case of an ABO-incompatible kidney transplantation in which preoperative DFPP failed to reduce the anti-ABO antibody titer to below 1:16. In this case, the kidney transplantation was performed after the completion of a preconditioning regimen consisting of infusions of an anti-CD20 monoclonal antibody (rituximab), DFPP, and a splenectomy.The patient was a 22-year-old man with Alport syndrome, who had been receiving dialysis treatment for 17 months. Because the patient’s blood type was O, and the donor’s was A (A1), three sessions of DFPP were performed 2 months before the kidney transplantation. However, the patient’s anti-A antibody titer did not drop to below 1:16, so the kidney transplantation was postponed. To enable the ABO-incompatible kidney transplantation to proceed, the following protocol was applied. Rituximab was infused weekly at a dose of 375 mg/m2 for 4 weeks. After the first rituximab infusion, the CD19+ B cell count rapidly decreased to below 1% in the peripheral blood. Two days after the fourth rituximab infusion, splenectomy was performed. After the splenectomy, four sessions of DFPP were performed. During the course of DFPP treatment, the anti-A antibody titer gradually decreased and was below 1:16 on the day of the transplantation. The kidney transplantation was performed by the standard method. The kidney allograft was performed immediately after the transplantation. No signs of humoral rejection were observed after revascularization. After the operation, the patient showed no signs of humoral rejection, and his serum creatinine levels were between 1.7 and 2.0 mg/dL. The anti-A antibody titer remained below 1:16 throughout the recovery period. A biopsy specimen obtained on postoperative day 12 showed no evidence of antibody-mediated rejection. After 3 months of follow-up, the kidney allograft continued to function well.A preconditioning regimen consisting of rituximab infusions and a splenectomy is a useful new strategy for performing ABO-incompatible kidney transplantations when the conventional preconditioning regimen does not work.