Pleural effusions usually result in a diagnostic dilemma. Pleural tuberculosis and lung cancer are pathologies that often give rise to exudates in humans. Several biochemical parameters are analyzed in exudates; however these parameters have limited efficiency for diagnostic separation of pleural tuberculosis and lung cancer. The objective of this study was to analyze the hyaluronan and sulphated glycosaminoglycans contents in pleural fluids and assessed their potential for discriminating between pleural tuberculosis and lung cancer. Pleural fluid samples originating from pleural tuberculosis and lung cancer were collected by thoracocentesis. Glycosaminoglycans were extracted by proteolyses, trichloroacetic acid and precipitation with ethanol. These glycosaminoglycans were analyzed for electrophoresis in agarose gel and enzymatic digestions with specific lyases, and they were quantified for carbazole reaction and densitometry. Hyaluronan in pleural fluids was quantified by the sandwich fluorescent ELISA-like assay. Molecular masses of glycosaminoglycans were determined by electrophoresis in polyacrylamide gel. Hyaluronan and chondroitin suphate were found in pleural tuberculosis and lung cancer samples, while dermatan sulphate and heparan sulphate were showed only in pleural tuberculosis samples. The hyaluronan and total glycosaminoglycans concentrations were highest in tuberculosis pleural. Concentrations, types and ratio of the glycosaminoglycans and molecular weights of hyaluronan were able to discriminate pleural tuberculosis from lung cancer. Hyaluronan content, such as sulphated glycosaminoglycans content, can be useful for the diagnostic separation of pleural tuberculosis and lung cancer.