We evaluated the usefulness of the tight-junction associated protein Claudin 4 (CL-4) in the diagnosis of mesothelioma and mimickers, analyzing biopsies from 454 tumors, including 82 mesotheliomas, 336 carcinomas of different origin (278 primary, 58 metastatic to serosae), 36 nonepithelial spindle cell neoplasms, as well as 97 cytological samples from reactive effusions (12), mesothelioma (23) and metastatic carcinomas (62). CL-4 was consistently negative in normal and reactive mesothelium, as well as in all 82 mesotheliomas. In contrast, strong reactivity was found in 57/58 serosal metastasis, and in 245/278 primary carcinomas, with uppermost expression (150/153) in those most frequently involved in the differential with mesothelioma (lung, breast, gastrointestinal tract, pancreas, ovary, primary serous papillary carcinoma of peritoneum). On effusions, reactive and neoplastic mesothelial cells were regularly negative, while metastatic tumor cells stained positively in 60/62 (96.8%) cases. Among spindle cell neoplasms, only 2/9 biphasic synovial sarcomas and 4/4 follicular dendritic cell sarcomas stained positively. Results indicate that CL-4 reacts with the majority of epithelial neoplasms that often metastasize to serous membranes, representing a pancarcinoma marker with extremely high sensitivity and specificity. CL-4 may be considered a primary immunohistochemical reagent to rule out the diagnosis of mesothelioma.