An assay using a phage-derived ligand to capture Escherichia coli O157:H7 prior to antibody detection was evaluated for assay specificity. Analysis of 200 strains showed that the assay was highly specific for the O157 serogroup. It detected all the O157:H7 strains including Shiga toxin-producing O157 nonmotile strains as well as O157 non-H7 strains. In addition, the assay detected various O157:H7 phenotypic variants that are not easily detected by routine analytical methods, as well as a rough strain that did not express O157 antigen and therefore is undetectable serologically. The phage ligand assay showed no cross-reactivity to the other E. coli serotypes. Isolates of Salmonella group N and a few Citrobacter freundii strains that cross-reacted with anti-O157 sera also showed cross-reactivity with the phage ligand. However, other strains that cross-reacted serologically with anti-O157 sera were correctly identified as negative with the phage ligand assay, including several strains of E. coli that nonspecifically autoagglutinate latex reagents.