Aims: The mechanism of the inactivation of Bacillus subtilis spores by reciprocal pressurization (RP) was unclear. Therefore, the mechanism was investigated. Methods and Results: To investigate the effects of RP and continuous pressurization (CP) treatments on the inactivation and injury of B. subtilis spores, spores were treated at 25, 35, 45 and 55degreesC under 200, 300 and 400 MPa. RP treatment was effective in injuring and inactivating spores. Scanning electron microscopy and transmission electron microscopy observation showed that spores treated by RP treatment were more morphologically and structurally changed than the ones treated by CP treatment. There were significant differences between the release of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by RP and CP treatments. From this result, it was concluded that the core fraction was released into the spore suspension. Conclusions: The mechanism of RP treatment is believed to work as follows: hydrostatic pressure treatment initiated germination of bacterial spores, and the repeated rapid decompression caused disruption, injury and inactivation of the germinated spores. Significance and Impact of the Study: This study indicated that the physical injury of bacterial spores was effective to inactivate the bacterial spores through the disruption of spores and leakage of their contents.