A method for the direct quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, in bakery products was developed. The analysis was based on robotized extraction followed by quantitation of cereulide by liquid chromatography-mass spectrometry and an assay of toxicity by the boar sperm motility inhibition test. The bioassay and the chemical assay gave comparable results, demonstrating that the extracted cereulide was in a biologically active form. Cereulide was formed when cereulide-producing B. cereus strains were present at > or = 106 CFU/g in products with water activity values of > 0.953 and pHs of > 5.6. Rice-containing pastries accumulated high contents of cereulide (0.3 to 5.5 microg/g [wet weight]) when stored at nonrefrigeration temperatures (21 to 23 degrees C). Cereulide was not formed in products stored at refrigeration temperatures (4 to 8 degrees C). Cereulide is not inactivated by heating during food processing. Therefore, direct analysis of this toxin in food is preferable to cultivating methods for assessing the risk of food poisoning by emetic B. cereus.