Pseudouridine in urine and plasma has been proved to be a useful tumour marker in many malignant conditions. We studied its usefulness in pleural fluid for distinguishing malignant from non-malignant pleural effusions. Pleural fluid pseudouridine concentrations in different groups of patients with pleural effusion (31 malignant, 29 benign, 16 unknown, 1 double pathology) was measured and compared. Its usefulness in distinguishing malignant from non-malignant pleural effusions was analysed by receiver-operating characteristic (ROC) curve analysis. Pseudouridine concentrations in the malignant group were significantly higher than the non-malignant group (P < 0.017, Bonferroni adjustment) with values overlapping extensively at the lower end. The area-under-the curve (AUC) value in the ROC curve analysis was 0.675 (P < 0.05). We conclude that the pleural fluid pseudouridine is of limited clinical value in distinguishing malignant from non-malignant pleural effusion due to its extensive overlap. However, it is useful when the concentration is higher than 65 mumol/L, which indicates malignancy.