Alteration of cell membrane permeability is speculated to be one of the mechanisms by which microwave kills microorganisms. It has been reported that permeability alteration may be reflected by cell shape changes observed under electron microscopy, or detected by measuring the leakage of intracellular protein and DNA using spectrophotometry. These methods, however, suffer from accuracy and sensitivity. Calcium is an important cell signaling molecule. Its level is tightly regulated with an intracellular to extracellular differential of approximately 1 to 10,000. Damage of cells will lead to alterations in membrane permeability and consequently influx of extracellular Ca2+. In the present study two probes, fluorescein diacetate (FDA) and fluo-3/AM, were used to quantify membrane permeability of E. coli and S. aureus after microwave treatment. These chemical probes, after metabolized by intracellular esterases and binding to Ca2+, emit strong fluorescence. Our data showed 20.7%, 28.1%, 74.8% and 89.8% increases in cel membrane permeability of E. coli after 50, 55, 60 and 65 degrees C microwave treatment, respectively, compared to untreated controls. Modest membrane permeability increases of 4.1%, 6.0%, 21.9% and 19.7% were seen for S. aureus. The permeability levels correlate with the fatality rates of microbes. These results suggest that alteration in cell membrane permeability contributes, in part, to the nonthermal-effect of cell killing by microwave.