Mesothelin has been proposed as a useful tool in the diagnosis of malignant pleural mesothelioma (MPM). We aimed to examine its diagnostic utility and the impact of renal impairment on results.We prospectively recruited 230 patients with new undiagnosed pleural effusions, testing serum (n=216) and pleural fluid (PF) (n=206) mesothelin (by ELISA) during the initial consultation.28/230 (12%) patients had MPM. Serum mesothelin gave sensitivity 59.3%, specificity 64.7%, Negative predictive value (NPV) 91.2%, Positive predictive value (PPV) 20.5% and PF, sensitivity 72.0%, specificity 87.5%, NPV 95.5%, PPV 46.2% for distinguishing effusions due to MPM. In a matched comparison, diagnostic characteristics of PF mesothelin were superior to serum (P=0.0001). Serum mesothelin levels in patients without MPM were higher in patients with renal impairment (p=0.007) while PF levels were unaffected. 19/35 (54%) patients with a benign pleural effusion and EGFR≤ 59 mL·min(-1) had a false positive serum mesothelin result.The diagnostic accuracy of pleural fluid mesothelin is superior to that of serum and is unaffected by renal function. In patients with a low pre-test probability of mesothelioma, a negative mesothelin test could be reassuring, because of its high NPV. Routine use of mesothelin testing in undiagnosed pleural effusions at presentation appears unhelpful.