Inactivation of bacterial endospores in food requires a combination of pressure and moderate heat. Endospore resistance of seven Clostridium botulinum strains was compared with those of Bacillus spp. (B. cereus, B. subtilis, B. licheniformis, B. smithii, B. amyloliquefaciens) and Thermoanaerobacterium thermosaccharolyticum with respect to pressure (600 to 800 MPa) and temperature (80 to 116 degrees C) treatments in mashed carrots. A large variation was observed in the pressure resistance of C. botulinum spores. Their reduction after treatments with 600 MPa at 80 degrees C for 1 s ranged from more than 5.5 log units to no reduction. Spores of the proteolytic C. botulinum TMW 2.357 exhibited a greater resistance to pressure than spores from all other bacteria examined, with the exception of B. amyloliquefaciens. Heat resistance of spores did not correlate with pressure resistance, either within strains of C. botulinum or when C. botulinum spores were compared with spores of T. thermosaccharolyticum. A quantitative release of dipicolinic acid was observed from C. botulinum spores on combined pressure and temperature treatments only after inactivation of more than 99.999% of the spores. Thus, dipicolinic acid is released by a physicochemical rather than a physiological process. The resistance of spores to combined pressure and temperature treatments correlated with their ability to retain dipicolinic acid. B. amyloliquefaciens, a mesophilic organism that forms highly pressure-resistant spores is proposed as a nonpathogenic target organism for high-pressure process development.