Escherichia coli O157:H7 is a human pathogen that causes food-born infections with symptoms that range from mild diarrhea to hemorrhagic colitis, and in a small percentage of patients, hemolytic uremic syndrome (HUS). Although tissue adherence is a critical factor in its pathogenicity, it has not been completely defined. The formation of biofilms is a mechanism that confers bacteria several advantages when growing under less favorable conditions. Biofilm development, structure, and regulation on abiotic surfaces have been extensively studied for E. coli O157:H7, but its role during pathogenesis or colonization of hosts still remains to be fully understood, mainly because most strains have not been shown to produce biofilms under normal laboratory culture conditions. However, an important linkage between biofilm formation and cellular adherence in E. coli O157:H7 strain EDL933 has been established. In this study we investigated the extent to which biofilm formation contributes to adherence in strains Sakai and 86-24 that lack in vitro biofilm forming activities. We determined that these poor biofilm forming strains are induced to produce biofilms in vitro under growth limiting conditions. We also deleted 6 genes in both strains related to biofilm formation in strain EDL933 (aroC, ecpD, hlyD, csgA,relA and yaiH). A significant reduction in adherence to HEp2 and T84 cells was demonstrated for all of the biofilm gene negative mutants, providing evidence that biofilm related genes found in EDL933 are also essential for cell adherence in Sakai and 86-24. Biofilm induced strains also adhered better to the epithelial cells. Finally, a microarray analysis showed that Sakai upregulates curli associated genes in the presence of cultured epithelial cells. These studies show that the adherence of E. coli O157:H7 to epithelial cells is strongly linked to biofilm formation, and that at least the initial steps in biofilm formation play important roles in the cellular adherence of E. coli O157:H7. This linkage might be further explored to understand how biofilm production at the mucosal epithelium interface contributes to high persistence and intermittent shedding in cattle and plays a role in the cellular adherence to human intestines and contribute to its pathogenicity there.