Enterohaemorrhagic Escherichia coli (EHEC) cause haemolytic uraemic syndrome (HUS), a thrombotic microangiopathy resulting from endothelial injury in the renal glomeruli and other organs. EHEC virulence factors that damage the microvascular endothelium play therefore major roles in the pathogenesis of HUS. We identified an EHEC strain that vacuolates and kills primary human glomerular microvascular endothelial cells (GMVECs) and a human brain microvascular endothelial cell (HBMEC) line. Because the vacuolating effect closely resembles those elicited on other cells by the vacuolating cytotoxin of Helicobacter pylori (VacA), we designated the factor responsible for this effect EHEC vacuolating cytotoxin (EHEC-Vac). EHEC-Vac (a secreted non-serine protease protein) binds to HBMECs rapidly and irreversibly, vacuolates within 30 min after exposure and the effect is maximally apparent at 48 h. Despite the vacuolisation, HBMECs survive for several days before they undergo necrosis. Electron and immunofluorescence microscopy demonstrate that the vacuoles induced by EHEC-Vac originate from lysosomes. Accordingly, they stain with neutral red indicating an acidic microenvironment. Similar to VacA, the EHEC-Vac-mediated vacuolisation is both prevented and reverted by the vacuolar proton pump inhibitor bafilomycin A1, suggesting a similar mechanism of vacuole formation by these toxins. Despite the similarity of phenotypes elicited by EHEC-Vac and VacA, genomic DNA from the EHEC-Vac-producing strain failed to hybridise to a vacA probe, as well as to probes derived from presently known E. coli vacuolating toxins. Through its microvascular endothelium-injuring potential combined with the ability to induce interleukin 6 release from these cells EHEC-Vac might contribute to the pathogenesis of HUS.