This study presents evidence that the pattern (diffuse or aggregative) of adherence of clinically relevant non-O157 Shiga toxin-producing Escherichia coli (STEC) to bovine recto-anal junction squamous epithelial cells is similar to that of E. coli O157, although the mechanisms of adherence appear to be distinct. Our results further suggest that novel adhesins, and not Intimin, are likely involved in non-O157 STEC adherence to bovine recto-anal junction squamous epithelial cells. These findings have important implications for the development of efficacious modalities for blocking adherence of non-O157 STEC to bovine gastrointestinal epithelial cells.