Aims: To determine the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide. Methods and Results: Killing of spores of B. subtilis with hydrogen peroxide caused no release of dipicolinic acid (DPA) and hydrogen peroxide-killed spores were not appreciably sensitized for DPA release upon a subsequent heat treatment. Hydrogen peroxide-killed spores appeared to initiate germination normally, released DPA and hydrolysed significant amounts of their cortex. However, the germinated killed spores did not swell, did not accumulate ATP or reduced flavin mononucleotide and the cores of these germinated spores were not accessible to nucleic acid stains. Conclusions: These data indicate that treatment with hydrogen peroxide results in spores in which the core cannot swell properly during spore germination. Significance and Impact of the Study: The results provide further information on the mechanism of killing of spores of Bacillus species by hydrogen peroxide.