The relationship between membrane permeabilization and loss of viability by pulsed electric fields (PEF) depending on the treatment intensity and the treatment media pH in two gram-positive (Lactobacillus plantarum, Listeria monocytogenes) and two gram-negative (Escherichia coli, Salmonella senftenberg 775W) bacterial species has been investigated. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI). Non-permanent/reversible permeabilization was detected when cells stained with PI during PEF resulted in higher fluorescence than that measured in cells stained after PEF. Whereas loss of viability of the two gram-negative bacteria was correlated with the sum of non-permanent and permanent membrane permeabilization when treated at pH 7.0, in the case of the two gram-positives, loss of viability was correlated with a permanent loss of membrane integrity. At pH 7.0, the four bacteria exhibited reversible permeabilization. However, whereas the gram-positives capable of reversing permeabilization survived, the gram-negative cells died, despite their capacity to reverse permeabilization immediately after PEF. Thus, resealing is not necessarily related to the survival of PEF-treated cells. In contrast, when cells were PEF-treated at pH 4.0 a more complicated picture emerged. Whereas loss of viability was correlated with a permanent loss of membrane integrity in L. monocytogenes cells, in L. plantarum the degree of permeabilization was higher, and in the gram-negative strains, much lower than the percentage of inactivated cells. These results support the view that membrane permeabilization is involved in the mechanism of bacterial inactivation by PEF, but the nature of membrane damage and its relationship with cell death depends on the bacterial species and the treatment medium pH.