A major virulence determinant of enteropathogenic Escherichia coli (EPEC) is the Tir molecule that is translocated into the plasma membrane where it orchestrates cytoskeletal rearrangements. Tir undergoes several phosphorylation events within host cells, with modification on a tyrosine essential for its actin-nucleating function. The EHEC (serotype O157:H7) Tir homologue is not tyrosine phosphorylated implying that it uses an alternative mechanism to nucleate actin. This is supported in this study by the demonstration that EHEC Tir is unable to functionally substitute for its EPEC homologue. Like EPEC, the EHEC Tir molecule is phosphorylated within host cells, with the actin-nucleating dysfunction correlated to an altered modification profile. In contrast to EHEC Tir, the EPEC Tir molecule mediated actin nucleation whether delivered into host cells by either strain. Thus, it would appear that EHEC encodes specific factor(s) that facilitate the correct modification of its Tir molecule within host cells. Domain-swapping experiments revealed that the N-terminal, alpha-actinin binding, Tir domains were functionally interchangeable, with both the actin-nucleating dysfunction and altered modification profiles linked to the EHEC C-terminal Tir domain. This tyrosine-independent modification process presumably confers an advantage to EHEC O157:H7 and may contribute to the prevalence of this strain in EHEC disease. The presented data are also consistent with EPEC and EHEC sharing non-phosphotyrosine phosphorylation event(s), with an important role for such modifications in Tir function. An EHEC-induced phosphotyrosine dephosphorylation activity is also identified.