Type 1 pili, characterized by mannose-inhibitable agglutination of fowl or guinea pig erythrocytes, have been found throughout the family Enterobacteriaceae. A radiolabeled probe was prepared from a restriction endonuclease-digested fragment of the Escherichia coli pil operon and used to detect homologous DNA sequences in 236 bacteria representing 11 genera of Enterobacteriaceae. Only isolates identified as E. coli or Shigella spp. exhibited homology. In contrast, mannose-sensitive hemagglutination was observed in nine genera. Probe DNA did not hybridize to plasmid DNA, indicating a chromosomal location for the pil operon. Analysis of restriction nuclease-digested whole-cell DNA from 60 E. coli and two Shigella sp. isolates indicated that internal sequences were conserved in most strains, but that changes in flanking sequences in the chromosome were common.