Escherichia coli O157:H7 is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a Type III secretion system, a mechanism shared by other enterohemorrhagic E. coli (EHEC). We recently reported that vaccination of cattle with Type III-secreted proteins (TTSPs) resulted in decreased shedding of the organism following both experimental infection as well as under conditions of natural exposure. In order to extend this to non-O157 EHEC serotypes, we examined the serological cross reactivity of TTSPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Western blotting experiments with polyclonal antisera directed against serotype O157:H7 TTSPs suggested that there was significant cross reactivity, although there was limited cross reactivity when two Tir- and EspA-specific monoclonal antibodies were used. Groups of cattle were then vaccinated with TTSPs produced from each of the above serotypes and the magnitude and specificity of the responses were measured. All animals responded well with antibodies to TTSPs of the homologous serotype. However, limited cross reactivity was observed against the others. No cross reactivity was observed against Tir and EspA of serotype O157:H7. These results suggest that vaccination of cattle with TTSPs as a means of reducing the risk of EHEC transmission to humans will induce protection that is serotype specific.