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Pulsed electric field (PEF) inactivation is a very effective method to kill bacteria in liquid while avoiding thermal damage. However, only a limited inactivation effect on spores has been reported to date and the possible mechanisms are still unclear. Therefore, a study of inactivation of spores using PEF in a pressurized flow system is reported in this paper. PEF with a maximum magnitude higher than 110 kV/cm and a pulse width of 100 ns has been applied to a carefully designed treatment chamber through which a suspension fluid of 0.5 MPa continuously flows. Using the proposed PEF inactivation method, maximum 6.7 log reductions were achieved for B. subtilis spores that were investigated. These reductions were much greater than those obtained by a heat inactivation approach. Through frequency analysis using the frequency components of the applied pulses and the frequency response of the equivalent circuit of the spore, it was found that most voltage is applied to the outside of the core in the lower frequency and to the inside in the upper frequency. Also, transmission electron microscope micrographs of B. subtilis spores were taken in order to verify the effect of the PEF treatment.