To elucidate factors influencing the sensitivity of Bacillus subtilis spores to DNA damaging chemicals. Wild-type spores of B. subtilis made at lower temperatures were more sensitive to the DNA damaging chemicals formaldehyde and nitrous acid than were spores made at higher temperatures, but this was not the case with the DNA alkylating agents ethylmethanesulphonate and methylmethanesulphonate. Spores lacking most DNA protective alpha/beta-type small, acid-soluble proteins (termed alpha(-)beta(-) spores) made at lower temperatures were also more sensitive to killing through DNA damage by hydrogen peroxide than were alpha(-)beta(-) spores made at higher temperatures. The spore coat, whose composition varies significantly with sporulation temperature, played only a minor role in spore resistance to these DNA damaging agents. Spores made at lower temperatures exhibited higher permeability to the methylamine and germinated more rapidly with the surfactant dodecylamine than did spores made at higher temperatures. Treatment of spores with the oxidizing agent cumene hydroperoxide sensitized the surviving spores to all these DNA damaging agents. The fatty acid composition of the inner membrane of spores made at different temperatures differed significantly, but levels of unsaturated fatty acids in the inner membrane did not influence spore resistance to DNA damaging agents or the sensitization to such agents by prior treatment with cumene hydroperoxide. The higher rates of methylamine permeation across the inner membrane of spores made at lower temperatures and the greater sensitivity of wild-type spores made at lower temperatures to formaldehyde and nitrous acid and of alpha(-)beta(-) spores made at lower temperatures to hydrogen peroxide, all agents that must pass through the spore's inner membrane to damage DNA in the spore core, suggest that the permeability of the inner membrane is a significant factor influencing spore sensitivity to these agents. The sensitization of spores to DNA damaging chemicals by pretreatment with an oxidizing agent, a treatment that increases the permeability of the spore's inner membrane, and the more rapid dodecylamine germination of spores made at lower temperatures are consistent with this suggestion. The results in this communication provide new insight into the factors that influence the resistance of spores of Bacillus species to chemicals that kill spores by damaging spore DNA.