Enterohaemorrhagic Escherichia coli (EHEC) survives exposure to acute acid stress during gastric passage and progresses to colonize the large intestine. We previously reported that acid stress significantly increases host adhesion of EHEC O157 : H7 and is associated with a coincident upregulation of the expression of a putative adhesin gene, yadK. Further gene expression analysis now confirms that yadK is minimally transcribed under unstressed conditions and is significantly upregulated under acid stress. Immunoblotting with an anti-YadK polyclonal antiserum demonstrates that YadK protein is also upregulated after acid stress. Disruption of yadK results in loss of the acid-induced adhesion increase seen for wild-type EHEC to human epithelial cells in vitro and complementation in trans fully restores the acid-induced adhesion phenotype to the wild-type level. Significantly, no difference is observed in adhesion of the unstressed yadK mutant relative to wild-type, indicating that YadK does not play a role in adhesion of unstressed EHEC. Anti-YadK antiserum inhibits the acid-induced adhesion enhancement of EHEC but has no effect on adhesion of unstressed EHEC. There is no significant difference in the viability of either the unstressed or the acid-stressed yadK mutant relative to the similarly treated wild-type, suggesting that yadK is not involved in acid tolerance. These results provide persuasive evidence that YadK plays a significant role in the adhesion of acid-stressed EHEC to epithelial cells, and support a role for acid stress as a factor which may regulate bacteria-host attachment and lead to increased EHEC colonization and virulence.