The aim of this work was to characterize adaptive mucosal immune responses to Escherichia coli O157:H7 at the principal site of colonization in the bovine species. Following experimental infection, extracts from terminal rectum mucosal samples were tested for IgA antibodies by immunoblotting against different bacterial antigens including: whole-cell E. coli O157:H7 with and without proteinase treatment, outer membrane and cytoplasmic preparations, secreted protein supernatants and purified E. coli O157 lipopolysaccharide and H7 flagellin. Lipopolysaccharide and H7 flagellin preparations were also used to coat enzyme-linked immunosorbent assay plates to determine mucosal IgG1 and IgA antibody titers. In this work, evidence is presented of strong local IgA immune responses induced following infection at the bovine terminal rectal mucosa directed against multiple antigens including type III secretion-dependent proteins, O157 lipopolysaccharide, H7 flagellin and OmpC.