Aim: To screen and characterize toxic, heat-stable substances produced by food borne strains from Bacillus subtilis group. Methods and Results: Using the boar sperm motility inhibition assay, six isolates from two outbreaks, out of the 94 isolates from 26 foods, were found to produce ethanol-soluble heat-stable substances that were toxic to sperm cells by depleting the mitochondrial membrane potentials. The toxic isolates were identified as Bacillus subtilis and B. mojavensis. Colon carcinoma cells (Caco-2) were used to model the contact with the human digestive tract. The extract of B. subtilis F 2564/96 depolarized the mitochondria in intact Caco-2 cells similarly as in sperm cells. The substance responsible for these effects was purified using HPLC and identified by electron spray ionization ion trap mass spectrometry analysis as amylosin. The temperature requirement for amylosin production was 21-37 degrees C for B. subtilis and 11-21 degrees C for B. mojavensis. Both species produced amylosin in air as well as in 7-8% CO2 with 8-9% O-2. Conclusions: Food borne illness related strains of B. subtilis and B. mojavensis, produced the heat-stable toxin amylosin. Significance and Impact of the Study: This is the first report that suggests a role for the heat-stable, ion-channel forming toxin amylosin, as a virulence factor in food borne Bacillus.