The Adhesive Factor/Rabbit 2 (AF/R2) is found in Escherichia coli strains of serovar O103:K-:H2 and rhamnose-negative biovars isolated from weaned rabbits with diarrhea. This adhesin allows the colonization of the distal parts of the digestive tract, a first step leading to severe inflammatory diarrhea and death of the animals. In vitro, AF/R2 expression mediates diffuse adhesion of E. coli on HeLa cells, adhesion to ileal villi of newborn and weaned rabbits and the presence of a major 32 kDa subunit in bacterial surface extracts. In this work, we constructed TnphoA mutants of the prototype strain B10 and selected an isogenic clone that did not express the AF/R2 32 kDa subunit when grown in permissive conditions in vitro. The pathogenicity of the wild type strain and of the isogenic mutant was compared by oral inoculation to 35-day-old weaned rabbits. The mutant showed impaired colonization and a highly significant loss of pathogenicity. However, the occurrence of residual weight losses, and of diarrheas and mortalities in some inoculated rabbits suggest that pathogenicity of rabbit O103 enteropathogeniclike E. coli (EPEC-like) strains is due to multiple virulence factors and that other virulence traits remain to be found.