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Exposures to nanosecond pulsed electric fields (nsPEFs) are known to induce intracellular responses, such as the induction of apoptosis, offering a promising new method to treat cancer. This and other secondary biological responses are believed to be promoted by an initial formation of nanopores in cellular membranes. The primarily responsible charging mechanisms depend on pulse duration and amplitude, as well as the conductivity of the extracellular medium. In comparison, the postexposure development of membrane integrity and secondary-cell responses depend on complex interaction of biophysical and biochemical processes. To assess the effect of exposure media beyond their electrical characteristics, we studied different exposure media with similar conductivities and osmolalities. Experiments were performed using a typical nsPEF regimen, as is used in apoptosis studies (eight pulses of 60-ns duration and 60 kV/cm). We investigated the development of membrane permeability with propidium iodide and cell survival with calcein-AM on biologically relevant times out to 20 or 90 min, respectively. We found a tenfold increase in permeabilization of the plasma membrane depending on the exposure medium and a similar effect on the cell viability. The results suggest that progression of membrane permeability and cell survival strongly depend on the composition of the extracellular medium, rather than its electrical characteristic alone.