Enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), make potent cytotoxins (Verotoxins [VT] or Shiga-like toxins), and possess a plasmid (approximately 60 megadaltons) that encodes a new fimbrial antigen and promotes attachment to epithelial cells. We evaluated the use of a DNA probe, prepared from a 3.4-kilobase segment of the EHEC plasmid, to detect EHEC. The probe hybridized with 106 (99%) of 107 O157:H7 and 34 (77%) of 44 O26:H11, VT-positive strains from patients with colitis, HUS, and diarrheal disease and hybridized with 21 (81%) of 26 VT-positive E. coli of serotypes other than O157:H7 or O26:H11 from patients with hemorrhagic colitis and HUS. We examined 601 other strains, including 18 serotype O26 isolates of H types other than H11, 306 enteropathogenic E. coli, 60 enteroinvasive E. coli, 119 enterotoxigenic E. coli, and 20 isolates from the urinary tract and 77 isolates from the normal intestinal flora; only one (O127:H-) was positive (specificity, 99.8%). Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC.