Several tumor markers have been evaluated in pleural fluid, but their clinical role has not been firmly established. The aim of this study is to determine the diagnostic value of carbohydrate antigen 549 (CA 549) levels in pleural fluid, and to compare it with another previously studied tumor markers: carcinoembryonic antigen (CEA), CA 15.3 and CA 72.4. We prospectively studied 252 patients with pleural effusion: 101 malignant (20 mesothelioma) and 151 of several benign diseases. The levels of the tumor markers were measured by immunoradiometric assays (RIA). CA 549 in pleural fluid has an acceptable sensitivity (0.49), with high specificity (0.99). The best combination of tumor markers for differentiating malignant from benign effusions was CA 549+CEA+CA 15.3, with a sensitivity of 0.65, specificity of 0.99 and accuracy of 0.85. The addition of any one tumor marker assay consistently improved the diagnostic value of cytology. In our study, none of the tumor markers was organ-specific. When mesothelioma and hematological malignancy were ruled-out, the combination of CA 549+CEA+CA 15.3, improved the results up to a sensitivity of 0.77, specificity of 1 and accuracy of 0.92. In conclusion, CA 549 assay has an acceptable sensitivity with high specificity. The best combination of tumor markers in this series with a high relative frequency of mesothelioma and low frequency of breast carcinoma was CA 549+CEA+CA 15.3. Individual tumor markers or their combination increased the sensitivity of pleural cytology.