Objective: Claudin-3 and claudin-4 have recently been reported as promising targets for the detection and diagnosis of cancer. This study was designed to evaluate the diagnostic value of claudin-3 and claudin-4 immunoreactivity to distinguish metastatic adenocarcinoma cells (MAC) from reactive mesothelial cells (RMC) in effusions. Study Design: Claudin-3 and claudin-4 immunocytochemical staining was performed on 234 cell block specimens, including 194 malignant effusions with MAC and 40 benign effusions with RMC. Any degree of membranous staining was considered positive. Results: Claudin-3 was positive in 190 (97.9%) out of 194 cases with MAC and in 3 (7.5%) out of 40 cases with RMC. Claudin-4 immunoreactivity was seen in all 194 (100%) cases with MAC and in 11 (27.5%) out of 40 cases with RMC. In all claudin-3- or claudin-4-positive RMC samples, the area of positive staining was <25% of the cells. Claudin-3 and claudin-4 efficiently discriminated between MAC and RMC (p < 0.001 for both), and claudin-3 was more specific than claudin-4 in differentiating between MAC and RMC (p < 0.05). Conclusion: These results suggest that claudin-3 and claudin-4 are good candidates to be included as MAC markers in the panel of antibodies to distinguish MAC from RMC in effusion specimens.