Aims: To determine the responses of spores of Bacillus subtilis and Bacillus anthracis surrogate Bacillus thuringiensis Al Hakam to I-2 treatment. Methods and Results: Spores of B. subtilis and B. thuringiensis killed by aqueous 30 degrees C-I-2 could germinate, and their inner membrane (IM) was intact. Spore coats were important in I-2 resistance, DNA-protective proteins were not important, and survivors of I-2 treatment were not mutagenized. Viabilities of I-2-treated, 90-98% killed spores were much lower on high-salinity media, and the treated spores were more heat sensitive than the untreated spores. Germinated I-2-killed spores were dead as determined by staining with nucleic acid dyes, and many appeared to have been lysed. Conclusions: Aqueous I-2 appeared to kill B. subtilis and B. thuringiensis spores such that spores lyse soon after they germinate, and not by causing DNA damage or rupture of spores' IM. I-2 treatment also generated many damaged spores that could only be recovered under nonstressful conditions. Significance and Impact of the Study: This work shows that spores of the model organism B. subtilis, and B. thuringiensis, a surrogate for B. anthracis spores, exhibit similar mechanisms of resistance to and killing by I-2. Generation by I-2 treatment of conditionally dead spores indicates that appropriate media are essential to efficiently enumerate viable I-2-treated spores.