Aims To investigate effects of the cationic surfactant cetyltrimethylammonium bromide (CTAB), a disinfectant, on spores of Bacillus species. Methods and Results The ability of CTAB to trigger release of Bacillus spores' large depot of dipicolinic acid (DPA) in a 1 : 1 chelate with Ca2+ (CaDPA), and to kill spores was investigated. CTAB-triggered CaDPA release from spores of Bacillus subtilis, Bacillus cereus and Bacillus megaterium, but was not followed by completion of germination. CaDPA release triggered by CTAB increased at higher temperatures, and was optimal for B. subtilis spores at pH 9 center dot 4 and 30 mu g ml(-1) CTAB. CTAB also killed Bacillus spores as shown by plate counts and vital staining of treated dormant spores, and after their germination. However, B. cereus and B. megaterium spores were more CTAB-sensitive than were B. subtilis spores. CaDPA release from and killing of CTAB-treated spores of isogenic B. subtilis mutants lacking germination proteins was also examined, and compared with effects of the well-known germinant dodecylamine on spores, to determine how CTAB exerts its effects on spores. Conclusions The results of this investigation showed that CTAB kills spores of three Bacillus species, perhaps by damaging the spore inner membrane, although it is also possible that some killing by this agent follows its triggering of spore germination. Significance and Impact of the Study The results of this work indicate that CTAB is also a disinfectant, but also a sporicide, and may be a useful adjunct in spore decontamination, especially at higher temperatures.