Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health problem and are commonly acquired via contact with ruminant faeces. The serotypes that predominantly associated with human infection are O157:H7 and O26:H^–. These serotypes differ in their virulence for calves. The molecular mechanisms underlying the carriage and virulence of EHEC in the ruminant intestine are poorly understood. All animal experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986 and were approved by the local Ethical Review Committee. Histological analysis of tissues of experimentally inoculated conventional calves 4 days post-inoculation revealed extensive adherence of an EHEC O26:H^– isolate to the colonic epithelium. In contrast, EHEC O157:H7 was scarcely detected on the epithelium at the same site in age-matched animals, indicating that O157:H7 and O26:H^– EHEC may colonise the bovine host in distinct ways. To identify E. coli O157:H7 and O26:H^– genes that are required for intestinal colonisation of calves we have used signature-tagged transposon mutagenesis. To date, c. 600 O26:H^– (strain 193) and c. 1900 O157:H7 (strain EDL933) transposon mutants have been screened. The site of transposon insertion was determined in 62 O26 mutants and 79 O157 mutants that were absent or poorly represented in the faeces and/or colonic mucosa. Several mutants of each serotype were isolated with insertions in Locus of Enterocyte Effacement (LEE) genes encoding putative structural components of a Type III protein secretion system that is required for the formation of attaching and effacing lesions, indicating that the LEE-encoded Type III secretion system plays a key role in the colonisation of the bovine intestine by EHEC O26:H^– and O157:H7. Putative non-LEE-encoded Type III secreted proteins were also found to be required including NleD (O157), and homologues of PkgA (O26) and GogB (O26). Expression of type 1 fimbriae appears to be detrimental for persistence in vivo. Cytotoxins (ehxA, pssA and efa1) facilitate the colonisation of calves by EHEC O26:H^–, but were not detected in the O157 screen indicating that different EHEC serotypes may differ in their reliance on cytotoxins to colonise the intestines of calves. We have identified a putative fimbrial operon that is required for colonisation of the bovine intestine by both EHEC O26:H^– and O157:H7. Further colonisation was dependent on numerous of ‘housekeeping genes’ mainly coding for enzymes involved in central intermediary metabolism, in transport systems and cryptic phage or prophage related genes. Single strain calf challenges were performed with O157 escN, map and z2200 mutants, and with O26 escN, fimE and pssA mutants. Our results will shed light on the molecular basis of EHEC serotype tissue tropism and facilitate the development of strategies to control EHEC in ruminants.