Background: Serum mesothelin is a new biomarker for the diagnosis of mesothelioma. Mesothelioma patients commonly present with pleural effusions. To define the clinical utility of mesothelin quantification in pleural fluid, we assessed its additional value over pleural fluid cytology and its short-term reproducibility and reliability following pleural inflammatory processes, including pleurodesis. Methods: Mesothelin was quantified in 424 pleural fluid and 64 serum samples by ELISA. Fluid was collected prospectively from 167 patients who presented with pleural effusions for investigation. Serial pleural fluid samples were obtained from patients (n=33) requiring repeated drainage. Mesothelin levels were also measured in patients (n=32) pre- and post pleurodesis Findings: Pleural fluid mesothelin concentrations were significantly higher in mesothelioma patients (n=24) over those with metastatic carcinomas (n=67) and benign effusions (n=75): median (QR25-QR75) = 40.3 (18.3-68.1) vs. 6.1 (1.5-13.2) vs. 3.7 (0.0-12.4) nM respectively, p<0.0001. Mesothelin measurement was superior to cytological examination in the diagnosis and exclusion of mesothelioma (sensitivity 71% vs. 35%; specificity 89% vs. 100%; negative predictive value 95% vs. 82% respectively). In patients with 'suspicious' cytology, pleural fluid mesothelin was 100% specific for mesothelioma, and in cytology-negative effusions (n=105), offered a negative predictive value of 94%. Intra-individual reproducibility of pleural fluid mesothelin was excellent: mean(±SD) variation -0.15(±8.41) nM in samples collected within seven days from mesothelioma patients. Measurements remained reliable following pleurodesis and were not affected by the presence of bacteria. Conclusion: Pleural fluid mesothelin provides additional diagnostic value over cytological examination. Mesothelin measurements are reproducible and not affected by inflammatory pleural processes.