The objective of this study was to measure the concentrations of tumor necrosis factor-alpha (TNFalpha) in pleural and peritoneal effusions of different causes and to verify whether TNFalpha, alpha-1-antitrypsin (alpha1AT), and complement factors C3 and C4 can be used in the differential diagnosis of serous effusion. One hundred forty-five serous effusions of various origins were analyzed. TNFalpha, alpha1AT, and complement factors C3 and C4 concentrations were measured simultaneously in blood and serous effusion using commercially available methods. Serous effusions were classified as follows: 102 exudates and 43 transudates. All variables were found to have good diagnostic value in the differential diagnosis of serous effusion. In the stepwise discriminant analysis, four variables were selected, producing a significant discriminant function (p < 0.001). Their order of selection was alpha1AT effusion, C4 serum, TNFalpha-effusion, and C3 effusion. Combined use of these variables increased remarkably the diagnostic value (in diagnosing exudates versus transudates) giving sensitivity = 93.14%; specificity = 90.70%; positive predictive value = 95.96%; negative predictive value = 84.78%. Determination of TNFalpha, complement factors C3 and C4, and alpha1AT may be a significant parameter in the differential diagnosis of serous effusions, particularly in those patients with malignant disease. Moreover, the combination of them significantly increased their diagnostic power.