Enteropathogenic Escherichia coli (EPEC) express an inducible bundle-forming pilus (BFP) associated with the presence of the EPEC adherence factor (EAF) plasmid and localized adherence (LA) on HEp-2 cells. The cloned structural gene (bfpA) encoding BFP was found to be specific for EPEC, as homologous sequences were found only in EPEC and not in other enteropathogens. The bfpA probe was slightly more sensitive than the EAF probe; among EPEC strains with LA, the bfpA and EAF probes hybridized with 99% and 96% of the strains, respectively. Immunoblotting of whole cell lysates of BFP-positive, EAF-positive or -negative, and LA-positive or -negative EPEC strains revealed variations in the size (18,500-21,000) of the expressed structural subunit of BFP, suggesting differences in processing that may account for discrepancies between the bfpA, EAF, and LA. Because the bfpA probe consists primarily of sequences encoding an important EPEC virulence factor, in contrast to the unknown function of sequences contained in the EAF probe, the bfpA probe may be useful in epidemiologic studies to detect EPEC.