Aims To determine how the microbicide ceragenin-13 (CSA-13) kills Bacillus subtilis spores prepared on growth or sporulation media, and these spores' properties. Methods and Results Spores made on Luria broth (LB) growth or double-strength Schaeffer's-glucose (2xSG) sporulation plates found that spores made on LB plates have coat defects as evidenced by their lower hypochlorite resistance, faster germination with dodecylamine and slower germination with Ca2+-dipicolinic acid (CaDPA) than 2xSG plate spores. CSA-13 triggered CaDPA release from spores, an early step in germination, but only well at 70 degrees C and better with spores made on LB than on 2xSG plates. Approximately 90% of spores with elevated levels of SpoVA proteins that form a CaDPA release channel, released CaDPA with CSA-13 at 70 degrees C, and faster with spores made on LB than 2xSG plates. Levels of CSA-13 killing of spores made on LB and 2xSG plates were similar to levels of CaDPA release triggered by this agent. Conclusions CSA-13 kills bacterial spores, but only at high concentrations and temperatures, and is preceded by CaDPA release. Significance and Impact of the Study CSA-13 is not a direct sporicide as reported previously, but most likely germinates spores via activation of spores' CaDPA channel, albeit inefficiently, and then killing the germinated spores.