Fifty polytransfused patients were prospectively studied to determine the frequency of post-transfusion alloimmunization and its influence on the response to platelet transfusion. Platelet- and HLA-specific antibodies were detected by means of the standard and antiglobulin-augmented lymphocytotoxicity techniques (LCT), the platelet suspension indirect immunofluorescence test (PSIIFT), and monoclonal antibody immobilization of platelet antigens (MAIPA). HLA antibodies were detected in 13 patients (26%) (IgM = 6; IgG = 6; IgM + IgG = 1). The standard LCT was positive in 12 of these 13 patients. Complement-independent HLA antibodies, only detectable in the PSIIFT and the antiglobulin-augmented LCT, were documented in two patients and were associated with poor post-transfusion platelet recovery in the patient who could be evaluated. All the HLA antibodies were detected in the PSIIFT, while only four were detected in the MAIPA. Platelet-specific alloantibodies were found in two patients by means of PSIIFT or MAIPA and may have led to poor post-transfusion platelet recovery in one patient. Platelet autoantibodies were detected in two patients but were not associated with platelet refractoriness. Paraformaldehyde-dependent platelet antibodies were detected in 11 patients but were not associated with platelet refractoriness.