An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA). A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B. cereus from outbreaks of food-borne illness, were evaluated for toxin production. Eleven of the outbreak strains exhibited cytotoxicity, including all six B. cereus strains associated with diarrhoeal illness. Several other isolates of B. cereus, and its relatives B. anthracis, B. mycoides and B. thuringiensis, exhibited similar cytotoxicity. The other species showed no cytotoxicity, with the exception of certain B. subtilis strains. The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.