Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two-hybrid assay for intimin-Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions. Many of the mutations fell within a 50-residue region near the C-terminus of intimin. Alanine-scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition. Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin-Tir complex of enteropathogenic E. coli predicts that each of these four intimin residues lies at the intimin-Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir. Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.