Several studies have been published on the inactivation of bacterial spores by using high pressure processing in combination with heat. None of the studies investigated the effect of the packaging system or the pressurizing fluid on spore inactivation. The objective of this study was to select and validate an appropriate packaging system and pressure transfer fluid for inactivation of Clostridium botulinum spores by using high pressure processing in combination with thermal processing. Inactivation of spores packaged in three packaging systems (plastic pouches, cryovials, and transfer pipettes) was measured in two pressure test systems (laboratory-scale and pilot-scale) at 700 MPa and >105°C. Total destruction (>6.6-log reduction) of the spores packaged in the graduated tube part of transfer pipettes was obtained after processing for up to 10 min at 118°C and 700 MPa in both pressure test systems, compared with the spores packaged either in plastic pouches or cryovials. Reduction of spores packaged in plastic pouches was lowest (<4.8 log) for both pressure test systems when processed at the same conditions (i.e., 700 MPa and 118°C). Within the pilot-scale pressure system, increasing the process temperature from 118 to 121°C at 700 MPa for 10 min resulted in only a small increase in spore reduction (<5.1 log) for spores packaged in plastic pouches, whereas there were no recoverable spores for either of the other two packaging systems. Use of plastic pouches for packaging spores in inactivation kinetic studies could lead to erroneous conclusions about the effect of high pressure in combination with heat. BioGlycol is the pressure-heat transfer fluid of choice, as compared with Duratherm oil, to maximize the temperature response rate during pressurization within the laboratory-scale pressure test system.