We developed Mn-supplemented, Fe-supplemented , and Ca-supplemented spores of Bacillus subtilis 168 and ATCC9372 in the respective presence of manganese , ferric and calcium ions, and compared their resistance to vapor-phase hydrogen peroxide with that to aqueous phase hydrogen peroxide and heat. Mn-supplemented spores were the most sensitive to heat and hydrogen peroxide solution but was the most resistant to vapor-phase hydrogen peroxide at low relative humidities as compared with Fe-supplemented and Ca-supplemented spores of the two strains. In the vapor-phase hydrogen peroxide treatment , the spore resistance was affected by the relative humidity during the period for spore preparation and was lowest at a relative humidity of 33%. The removal of the spore coat from the spores of strain 168 markedly sensitized Fe-supplemented and Ca-supplemented spores , and also slightly sensitized Mnsupplemented spores, to vapor-phase hydrogen peroxide. These results indicate that both divalent cations in spores and the relative humidity affect the spore resistance to hydrogen peroxide vapor and suggest that the spore coat plays a role in this resistance.