Pathogenic F165-positive Escherichia coli isolates of porcine and bovine origin possess gene clusters related to extraintestinal E. coli fimbrial operons pap, sfa, and afaI. Probes from different segments of the pap, sfa, and afaI operons were used in Southern hybridization to analyze 18 F165-positive, mannose-resistant hemagglutinating E. coli isolates possessing pap- and sfa-, pap- and afa-, or pap-related sequences. Only single copies of the pap-, sfa-, or afa-related sequences were found among the isolates, and the position of each sequence with respect to those of adjacent sequences was variable. Expression of the P and F adhesins individually or concurrently was associated with the presence of a single copy of pap-related sequences. Gene clusters related to pap were structurally similar to the pap operon in certain isolates or the prs operon in others. sfa-related sequences showed few internal structural polymorphisms and were structurally similar to the foc rather than the sfa operon. afa-related sequences showed many internal structural polymorphisms compared with the afa-related sequences from prototype strain KS52. These results demonstrate that the pap- and sfa-related sequences in F165-positive isolates are closely related to the prototype pap operon and foc operon of the P family and Sfa family, respectively. afa-related sequences, on the other hand, display heterogeneity and differ from the prototype afaI operon.