Two cases of trimethoprim-sulfamethoxazole (TMP-SMX)-induced immune thrombocytopenia are reported in which unusual drug-dependent platelet antibodies were demonstrated by immunofluorescence and enzyme-linked immunosorbent assay. Whereas two distinct sulfamethoxazole-dependent antibodies of the IgG and IgM class were detectable in the serum of one patient, the serum of the other patient contained a platelet antibody exclusively reactive with N-4-acetyl-sulfamethoxazole, a metabolite of sulfamethoxazole. Urine from a healthy volunteer collected after administration of therapeutic doses of TMP-SMX proved to be an appropriate source of ex vivo metabolites for antibody testing. The results of this study stress the role of metabolite-specific antibodies in drug-dependent immune thrombocytopenia and underscore the necessity of including metabolite preparations of drugs in serologic analyses.