Enteropathogenic Escherichia coli (EPEC), a cause of childhood diarrhea, grow on the surface of the small intestine and on cultured epithelial cells as colonies of adherent bacteria. When propagated on solid medium containing blood or attached to HEp-2 cells, EPEC express ropelike bundles of filaments, termed bundle-forming pili (BFP), that create a network of fibers that bind together the individual organisms. BFP were found to be expressed by five EPEC serogroups, each harboring a ∼92-kilobase plasmid previously known to be important for virulence in humans. When two of these strains were cured of this plasmid, they neither expressed BFP nor grew as adherent colonies. An antiserum to BFP reduced the capacity of EPEC to infect cultured epithelial cells. BFP are composed of a repeating subunit of 19,500 daltons, the amino-terminal amino acid sequence of this subunit is homologous to that of the toxin-coregulated pilin of Vibrio cholerae.