A quantitative immunofluorescence PA[platelet-associated]-IgG assay was used to detect alloimmunity to platelets. The assay identified serum alloantibodies in 10 of 14 multitransfused patients and for 2 of 3 infants with neonatal thrombocytopenia. The correct separation of all multitransfused patients into alloimmune and nonalloimmune groups by the PA-IgG assay was substantiated with 51Cr-labeled platelet survival studies. The allogeneic nature of the serum antibodies was demonstrated by progressive absorption of the antibody with increasing numbers of allogeneic platelets, but not with autologous platelets. The PA-IgG assay sensitivity for detection of serum alloantibodies was superior to that of platelet aggregation [PAG], platelet serotonin release [PSR] and lymphocytotoxicity testing [LCT]. In dilution experiments with alloimmune serum, elevated levels of serum PA-IgG could still be detected on donor platelets when PAG and PSR tests became negative. Platelet survival studies with selected platelets performed in the 10 alloimmunized, multitransfused patients confirmed the results of the PA-IgG assays, predicting alloimmunity to the donor platelets. PAG, PSR and LCT indicated alloimmunity for 50% or less of the patients. Reduced platelet survival times were also seen with HLA A- and HLA B- matched donor platelets when donor-recipient incompatibility was demonstrated by the PA-IgG assay. Thus the PA-IgG assay provides a sensitive method to detect serum platelet alloantibodies and may offer a technique in platelet crossmatching.