Food poisoning laboratories identify Bacillus cereus using routine methods that may not differentiate all Bacillus cereus group species. We recharacterized Bacillus food-poisoning strains from 39 outbreaks and identified B. cereus in 23 outbreaks, B. thuringiensis in 4, B. mycoides in 1, and mixed strains of Bacillus in 11 outbreaks. (16,19,22). They are difficult to discern using standard biochemical schemes, chemotaxonomic methods, or phylogenetically relevant target genes (1, 2), and many distinguishing pathogenicity markers in this group can be attributed to mobile plasmids (18,21,22,23). B. cereus sensu stricto carries the plasmid-borne emetic toxin cereulide (ces) (7,13,14), and B. thuringiensis carries insecticidal crystal protein (ICP) (cry) genes on one or more plasmids (3, 5, 6). The differentiation of B. cereus group members using molecular techniques is not routine in food-poisoning diagnostic methods and may cause underreporting of species such as B. thuringiensis (1,8 DNA was extracted by lysing pure culture in a heating block at 102°C for 10 min. Microcentrifuged supernatant was frozen at Ϫ80°C until required. Pathogenicity genes for emetic cereulide toxin (nonribosomal peptide synthetase [NRPS]) and ICP (cry1 or cry2) were detected in multiplex PCR assays (7, 10) shown in Fig. 1. Each master mix contained 0.8 M of each primer, hot start master mix, diethyl pyrocarbonate water (20 l), and 5 l of DNA. The PCR products were loaded onto 2% agarose gels made with 0.5ϫ Tris-borate-EDTA buffer and ethidium bromide (1 g ml Ϫ1 ). The gels were electrophoresed at 120 V for 30 min and then visualized on a Bio-Rad Gel Doc 2000.Strains positive for NRPS were designated as B. cereus, those positive for ICP (by microscopy or PCR) as B. thuringiensis, those with rhizoidal growth on nutrient agar as B. mycoides, and all other strains with the typical B. cereus phenotype as B. cereus NRPS Ϫ ICP Ϫ . PCR-negative isolates were further examined for ICP crystals using transmission electron microscopy (TEM) since B. thuringiensis strains may carry one to six cry genes and there is no universal method available to detect all cry genes (there are currently more than 150 cry1 toxins) (17; Bacillus thuringiensis toxin nomenclature [http: //www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/]). Samples of B. cereus group bacteria were prepared for electron microscopy by fixation with 2% glutaraldehyde and 1% para-