The aim of this study was to assess changes in the acid-base equilibrium of pleural fluid during the first 2 hours after thoracentesis and to determine whether, as with arterial blood, it is important to keep the fluid on ice. A prospective, descriptive, comparative study was performed in 53 consecutive patients with pleural effusion. Thoracentesis was performed and pleural fluid was collected in 5 heparinized syringes to determine the pH, PO2, and PCO2 at baseline and at 30, 60, 90, and 120 minutes. In the first 26 patients, pleural fluid was collected in a further 4 syringes that were kept on ice prior to performing the same measurements at 30, 60, 90, and 120 minutes. The patients had a mean (SD) age of 70 (14) years, 66% were smokers, 72% were men, 63% had right-sided pleural effusion, 85% had unilateral effusion, and 15% had massive effusion. In 10 patients the effusion was a transudate, in 35 it was lymphocytic, and in 8 it was neutrophilic. The etiology was benign in 34 cases and neoplastic in 19 cases. The baseline pH was 7.35 (0.1) and baseline values of PO2 and PCO2 were 57.8 (20) mm Hg and 53.7 (15) mm Hg, respectively. No significant changes were observed in the first 2 hours for either pH or PCO2, whereas PO2 did undergo a significant change over this period. The difference between the baseline value and the value obtained at 120 minutes was 0.005 (0.02) for pH, 12.5 (19) mm Hg for PO2, and 0.8 (3) mm Hg for PCO2, with correlation coefficients of 0.97, 0.49, and 0.98, respectively. Comparison of values by simple regression analysis did not reveal a significant difference in the changes in pH, PO2, or PCO2 associated with keeping samples on ice. Multivariate analysis revealed that neoplastic effusion and a higher red blood cell count in pleural fluid had a significant influence on pH changes. The pH and PCO2 of pleural fluid did not change significantly during the first 2 hours following thoracentesis, whereas PO2 did undergo a significant change. Keeping samples on ice during this period is unnecessary. Only a higher red blood cell count in pleural fluid and neoplastic effusion had a limited effect on changes in the pH of samples from our patients during the first 2 hours following thoracentesis.