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Claudin-4 (CL-4) is a tight junction–associated protein that is expressed in most epithelial cells but absent in mesothelial cells. The purpose of this study is to evaluate the utility of CL-4 immunostaining for assisting in the differential diagnosis of mesothelioma. Sixty mesotheliomas (40 epithelioid, 10 biphasic, and 10 sarcomatoid), 185 carcinomas of different origins that can potentially be confused with mesotheliomas, 37 soft-tissue sarcomas, and 5 melanomas were investigated for CL-4 expression. All 60 mesotheliomas were CL-4 negative. In contrast, 169 (91%) of 185 carcinomas expressed this marker. Five of 8 desmoplastic small round cell tumors and the epithelial component of all 5 biphasic synovial sarcomas were CL-4 positive, whereas none of the remaining soft-tissue sarcomas or melanomas expressed this marker. It is concluded that CL-4 is a highly specific and sensitive immunohistochemical marker for assisting in distinguishing epithelioid mesotheliomas from metastatic carcinomas to the serosal membranes.