The association of the pap operon with the CS31A and F17 adhesins was studied with 255 Escherichia coli strains isolated from calves, lambs, or humans with diarrhea. The three classes of PapG adhesin with different receptor binding preferences were also screened. The pap operon was associated with 50 and 36% of human strains that produced CS31A and ovine strains that produced F17, respectively. Among the bovine isolates, the pap operon was detected in 61% of the CS31A-positive isolates and 72% of the strains that produce both CS31A and F17. The class II adhesin gene was present in bovine (20%) and ovine (71%) isolates. Both class II and III adhesins were genetically associated with 36% of the human strains. The highest prevalence of the pap operon was observed among E. coli strains that produce additional adhesins involved in the binding of bacteria to intestinal cells. Among the bovine isolates, the reference strain for CS31A and F17c was found to be positive for the pap operon. Phenotypic and genotypic characterizations were undertaken. Pap_31A appeared as fine and flexible fimbriae surrounding the bacteria but did not mediate adhesion to calf intestinal villi. Pap_31A production was optimal with bacteria cultured on minimal growth media and repressed by addition of exogenous leucine. The deduced amino acid sequence of the PapA_31A structural subunit showed 57 to 97% identity with the different P-related structural subunits produced by E. coli strains isolated from pigs with septicemia or humans with urinary tract infections. None of the three papG allelic variants was detected, but a homologous papG gene was present in the chromosome of strain 31A.