The identification of metastatic cells in serous effusions has prognostic and therapeutic implications, thus leading to a continuous search for improvement of the existing diagnostic procedures, including immunocytochemistry. To evaluate the usefulness of an antibody recognizing the tight junction-associated protein Claudin 4 in detecting metastatic tumor cells and in the differential with reactive and neoplastic mesothelium, we stained 345 cases of benign and neoplastic serous effusions obtained from pleura, peritoneum, and pericardium. Two-hundred and twenty-eight of 230 cases (99.1%) of epithelial metastasis of different origin were strongly stained by anti-Claudin 4, whereas all cases of reactive mesothelitis (78) and malignant mesothelioma (37) were negative. With the exception of a single case of ovarian carcinoma hypercalcemic-type, all tumors originating from the anatomical sites that most frequently metastasize to the serosae, including lung (61), breast (23), female genital tract (67), gastrointestinal tract (27), and peritoneum (6), were found to be positive. Claudin 4 was also extremely useful in detecting single-tumor cells dispersed among heavy inflammatory reaction. Because of its high sensitivity (99.1%) and specificity (100%), Claudin 4 might be used as an ideal "single-shot" marker for the identification of metastatic epithelial cells in serous effusions.