Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 (stx 1 ؉ stx 2 ؉ eae ؉ ) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type ␥. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.Shiga toxin-producing Escherichia coli (STEC), predominantly of serotype O157:H7, is now one of the most important etiologic agents in hemorrhagic colitis and hemolytic-uremic syndrome (6,7,8,12,15,30). The ability of STEC to cause serious disease in humans is related to the production of one or more Shiga toxins (stx 1 , stx 2 , or their variants), which inhibits protein synthesis of host cells, thus leading to cell death (13,20). STEC bacteria comprise a serologically diverse group of food-borne, zoonotic pathogens, of which those of serotype O157:H7 have been epidemiologically significant worldwide because they are notoriously associated with life-threatening disease (12). However, in some geographic areas, non-O157 strains are more commonly isolated from persons with diarrhea or hemolytic-uremic syndrome than are O157 STEC strains (25,28). Hemorrhagic colitis is caused by a number of serotypes of STEC (15). Antibodies to the O157 antigen are used in many assays to detect O157:H7 isolates in clinical and food samples. However, previous studies showed that the anti-O157 sera cross-reacted with Citrobacter freundii and other bacterial species (4, 24). Although detection of enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) by using a monoclonal antibody has been reported earlier (14), the development of monoclonal antibodies is expensive for many laboratories. Biochemical methods of identifying strains of EHEC, a subgroup of STEC, are based on biochemical markers such as sorbitol fermentation deficiency and -D-glucuronidase nonproductivity of the O157 serotype of E. coli (10, 21). The existence of sorbitol...