The utility of tumour markers (TM) in the differential diagnosis of cancer in serous effusion (fluid effusion (FE)) has been the subject of controversy. The aim of this study was to prospectively validate our previous study and to assess whether the addition of adenosine deaminase (ADA), C-reactive protein (CRP) or percentage of polymorphonuclear cells (%PN) allows the identification of false positives. In this study, carcinoembryonic antigen, cancer antigen 15-3, cancer antigen 19-9, ADA, CRP and %PN in FE were determined in 347 patients with 391 effusions. Effusions were considered as malignant effusion when at least one TM in serum exceeded the cutoff and the ratio FE/S was higher than 1.2. Also, cases with values of ADA, CRP and %PN above the established cutoffs in serous effusion were considered as potential false positives. The combined sensitivity and specificity of the three TM was 76.2 % (95 % confidence intervals (CI) 67.8-83.3 %) and 97.0 % (95 % CI 94.1-98.7), respectively. Subanalysis of the 318 cases with previous criteria and negative ADA, CRP and %PN obtained sensitivities of 78.4 % (95 % CI 69.4-85.6) and a specificity of 100 % (95 % CI 98.2-100). The results obtained validate our previous study and are improved with the addition of ADA, CRP and %PN. TM in serous effusions and serum could be useful for the diagnostic assessment of patients with serous effusions.