Aims: To determine the mechanism of killing of spores of Bacillus subtilis by orthophthalaldehyde (OPA), an aromatic dialdehyde currently in use as an antimicrobial agent. Methods and Results: OPA is sporicidal, although spores are much more OPA resistant than are vegetative cells. Bacillus subtilis mutants deficient in DNA repair, spore DNA protection and spore coat assembly have been used to show that (i) the coat appears to be a major component of spore OPA resistance, which is acquired late in sporulation of B. subtilis at the time of spore coat maturation, and (ii) B. subtilis spores are not killed by OPA through DNA damage but by elimination of spore germination. Furthermore, OPA-treated spores that cannot germinate are not recovered by artificial germinants or by treatment with NaOH or lysozyme. Conclusions: OPA appears to kill spores by blocking the spore germination process. Significance and Impact of the Study: This work provides information on the mechanism of spore resistance to, and spore killing by, the disinfectant, OPA.