Escherichia coli O157:H7 is known as an important cause of hemorrhagic colitis and hemolytic uremic syndrome. Real-time procedures that are sensitive for detecting small populations of this bacterium in food are lacking and needed. An expression library was constructed by ligation of BamHI-EcoRI DNA fragments of E. coli O157:H7 to plasmid vector pUC19 and transformation of recombinant plasmids to E. coli JM109. A clone that contained a specific DNA fragment of E. coli O157:H7 was identified by colony immunoblot assay using monoclonal antibody MAb 4E8C12 that uniquely links to E. coli O157:H7 and a few other serotypes of verotoxin-producing E. coli. The DNA sequence of the clone consisted of 110 bp of 5' region of enterohemorrhagic E. coli (EHEC) eae gene and a 688 bp DNA fragment adjacent to 5' end of the eae gene, including an unknown function gene encoding 156 amino acids. A pair of oligonucleotide primers was synthesized based on the sequence of the 688 bp fragment. The primers were used in a polymerase chain reaction (PCR) to amplify a target DNA of 633 bp. The primers amplified 1 ng of DNA from 67 strains of E. coli O157:H7, two strains of E. coli O157:NM, and 7 of 11 E. coli O55:H7 and O55:NM strains, but not 50 ng of DNA from 34 strains of 29 other E. coli serotypes and 25 strains of 8 other bacterial species. Annealing temperatures from 60 to 63 degrees C could be used for the PCR without loss of specificity. The minimum amount of target DNA detected by the PCR was 5 pg. When a boiling method and GeneReleaser were used, the PCR was able to detect as few as 25 and 38 CFU of E. coli O157:H7, respectively, in 3 h.