Background and aim: Pleural effusions (PE) are a common clinical entity resulting from pathologies that affect the pleural space such as congestive heart failure, malignancy and pneumonia. The osmolality of the pleural fluid has never been studied as well as the effects of its changes on the pleural membrane. The purpose of this study was to identify the osmolality levels of PEs of different etiologies and to assess the potential effects of osmolality imbalance on the pleural permeability. Materials and methods: We measured the osmolality of the PEs of 64 consecutive patients (6 with transudative, 11 with parapneumonic and 47 with malignant pleural effusions) that were hospitalized in the University Hospital of Larissa. Subsequently, we selected clinically relevant hyper- and hypo- osmolality levels and performed assessment of the permeability of sheep parietal pleura by means of Ussing chamber experiments. Results: The mean pleural fluid osmolality was 291.7 ± 24.89 mOms/Kg (95 % CI: 285.4–297.9), and it varied among the three groups of PEs (p = 0.05). Transformed osmolality values were associated with pH and glucose levels in the PEs. After exposure of the sheep parietal pleura to 240 mOsm/kg (hyposmolar) the transmesothelial resistance (RTM) significantly increased (p < 0.05) while at 340 mOsm/kg (hyperosmolar) the RTM was not significantly altered. Conclusions: PEs osmolality differs depending on the underlying pathology and is linked to PE pH and glucose. Hypo-osmotic PEs can lead to decreased pleural permeability. These results warrant further study of the PEs osmolality levels on the function of the pleural mesothelial cells.