This article investigates the inactivation mechanism of high-pressure food treatment, considered as alternative to conventional biocidal processes. We aimed to determine intracellular pH decrease under CO2 and N2O pressure, so far postulated as one of the main causes of inactivation. Working with a lab-scale bioreactor in mild conditions - 25 degrees C and pressures up to 8 MPa - we monitored - for the first time during pressurization - cytoplasmic pH variations of Listeria innocua labeled with pH-sensitive fluorophores based on fluorescein. We show that carbonic acid, due to solubilization of CO2 into the aqueous phase, causes a rapid pH drop in the cytosol, reaching pH 4.8 at 1 MPa and falls below the detection limit of the indicator fluorophore of pH 4.0. This correlates with a reduced viability (below 90%) in all the pressure ranges investigated. Contrarily, treatment under N2O pressure reduces cell viability without significant pH-drop neither of intra- nor extra-cellular liquid at any pressure investigated. The pH value remains between 7 and 6 while an inactivation of more than 80% is achieved at 8 MPa. Our data clearly demonstrate that, as a critical pressure is achieved, microbial inactivation is mainly due to pressure-induced membrane permeation - stimulated by non-acidifying fluids as well, rather then cytoplasmic acidification, as widely argued so far. A definitive understanding of the microbial inactivation mechanism due to CO2/N2O under pressure has been advanced significantly. (C) 2011 Elsevier B.V. All rights reserved.