Aims To determine how hydrated Bacillus anthracis spores are killed in a high-temperature gas environment (HTGE), and how spores of several Bacillus species including B.anthracis are killed by UV radiation, dry heat, wet heat and desiccation. Methods and Results Hydrated B.anthracis spores were HTGE treated at c. 220 degrees C for 50ms, and the treated spores were tested for germination, mutagenesis, rupture and loss of dipicolinic acid. Spores of this and other Bacillus species were also examined for mutagenesis by UV, wet and dry heat and desiccation. There was no rupture of HTGE-treated B.anthracis spores killed 90-99 center dot 9%, no mutagenesis, and release of DPA and loss of germination were much slower than spore killing. However, killing of spores of B.anthracis, Bacillus thuringiensis and Bacillus subtilis by UV radiation or dry heat, but not wet heat in water or ethanol, was accompanied by mutagenesis. Conclusions It appears likely that HTGE treatment kills B.anthracis spores by damage to spore core proteins. In addition, various killing regimens inactivate spores of a number of Bacillus species by the same mechanisms. Significance and Impact of the Study This work indicates how hydrated spores treated in a HTGE such as might be used to destroy biological warfare agent stocks are killed. The work also indicates that mechanisms whereby different agents kill spores are similar with spores of different Bacillus species.