Membrane permeabilization, caused by pulsed electric field (PEF) processing of microbial cells, was investigated by measurement of propidium iodide (PI) uptake with flow cytometry. Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae was determined by viable counts, and leakage of intracellular compounds, such as ATP and UV-absorbing substances, was measured in the extracellular environment. Electrical field strength and pulse duration influenced membrane permeabilization of all three tested organisms of which S. cerevisiae was the most PEF sensitive, followed by E. coli and L. innocua. It was shown by viable counts, PI uptake and leakage of intracellular compounds that L. innocua was the most resistant. Increased inactivation corresponded to greater numbers of permeabilized cells, which were reflected by increased PI uptake and larger amounts of intracellular compounds leaking from cells. For E. coli and L. innocua, a linear relationship was observed between the number of inactivated cells (determined as CFU) and cells with permeated membranes (determined by PI uptake), with higher number of inactivated cells than permeated cells. Increased leakage of intracellular compounds with increasing treatment severity provided further evidence that cells were permeabilized. For S. cerevisiae, there was higher PI uptake after PEF treatments, although very little or no inactivation was observed. Results suggest that E. coli and L. innocua cells, which took up PI, lost their ability to multiply, whereas cells of S. cerevisiae, which also took up PI, were not necessarily lethally permeabilized. © 2004 Elsevier B.V. All rights reserved.