Monoclonal antibody (MAb) HBA71, which was raised against Ewing's sarcoma cells, recognizes a cell-surface glycoprotein, p30/32MIC2, that is encoded by the MIC2 gene in the pseudoautosomal region of human chromosomes X and Y. This immunohistochemical study evaluates the specificity and sensitivity of MAb HBA71 for tumor diagnosis. Frozen and paraffin-embedded tissues of more than 300 tumors of diverse histologic type, including more than 100 small round cell tumors of childhood and adolescence, were tested with this MAb by the avidin-biotin immunoperoxidase procedure. The authors found HBA71 immunoreactivity in 61 of 63 Ewing's sarcomas studied and 9 of 11 primitive neuroectodermal tumors and peripheral neuroepitheliomas. HBA71-negative tumors included neuroblastomas (0 of 24), melanomas (0 of 13), an esthesioneuroblastoma, small cell osteosarcomas (0 of 2), a malignant ectomesenchymoma, desmoplastic SRCT (0 of 5), and medulloblastomas (0 of 5). Heterogeneous expression of HBA71 immunostaining was found in some embryonal rhabdomyosarcomas (3 of 14) and astrocytomas (4 of 7), and in a few neuroendocrine tumors (4 of 26), carcinomas (3 of 94), and lymphomas (6 of 30). Because Ewing's sarcomas are consistently HBA71 positive, the authors searched for antigen-positive normal cells that may represent precursors for these tumors; however, no obvious candidate for the elusive cell of origin for Ewing's sarcoma was identified in the normal fetal tissues tested. Their findings indicate that HBA71 is a highly restricted cell-surface antigen of Ewing's sarcomas and primitive neuroectodermal tumors, and immunohistochemistry employing this antibody may be of value in the differential diagnosis of selected small round cell tumors in childhood and adolescence.