OBJECTIVE To determine the incidence and the rate of red blood cell alloimmunization in polytransfused patients. METHODS A polytransfused patient was defined as having received at least 6 units of red cell concentrates during a 3-month period. The records of all patients (n = 12,904) who had received red blood cell units were examined retrospectively by searching the computer database at Hospital Israelita Albert Einstein in São Paulo, Brazil, over a 6-year period, between 2003 and 2009. RESULTS During this time, 77,049 red cell concentrate transfusions were performed in 12,904 patients. There were 3,044 polytransfused patients, 227 of whom (7.5%) presented with irregular erythrocyte antibodies. The prevalence of alloantibody specificity was: Anti-E>anti-D>anti-K>anti-C>anti-Dia>anti-c>anti-Jka>anti-S in 227 polytransfused patients. We found combinations of alloantibodies in 79 patients (34.8%), and the most common specificities were against the Rh and/or Kell systems. These antibodies show clinical significance, as they can cause delayed hemolytic transfusion reactions and perinatal hemolytic disease. About 20% of the patients showed an IgG autoantibody isolated or combined with alloantibodies. Interestingly, a high incidence of antibodies against low frequency antigens was detected in this study, mainly anti-Dia. CONCLUSIONS Polytransfused patients have a high probability of developing alloantibodies whether alone or combined with autoantibodies and antibodies against low frequency antigens. Transfusion of red blood cells with a phenotype-compatible with RH (C, E, c), K, Fya, and Jka antigens is recommended for polytransfused patients in order to prevent alloimmunization and hemolytic transfusion reactions.