In this study, we consider the effect of carbon dioxide (CO2) on the intracellular and extracellular pH of a saline solution of a test-microorgan isms Bacillus subtilis. The cytoplasmatic pH was determined by means of a flow cytometry with the fluorescent probe 5(and 6-)-carboxyfluorescein ester (cFSE). The physiological suspension of cells with the addition of the probe was first exposed to high pressure CO2 for 5 min at different temperatures. The flow cytometry analysis indicated an intracellular depletion inside the cell caused by the action of CO2, down to 3, the depletion being dependent on inactivation ratio. In addition, the extracellular pH was determined theoretically by means of the statistical associated fluid theory equation of state (SAFT EOS): it was demonstrated that CO2 under pressure dissolves into liquid phase and acidifies the medium down to 3 at 80 bar and 303.15K. The results show a strong influence between extracellular and intracellular pH, and lead to the conclusion that a strong reduction of the pH homeostasis of the cell can be claimed as one of the most probable cause of inactivation of CO2 pasteurization. (c) 2005 Wiley Periodicals, Inc.