The ability of particular Escherichia coli strains to lyse erythrocytes of several mammalian species was first described by Kayser in 1903 (1 ). This phenomenon was termed hemolysis and the bacterial determinants involved were termed hemolysins . The best characterized E. coli hemolysin is α-hemolysin, which is mainly produced by uropathogenic E. coli (UPEC). When blood agar plates are inoculated with α-hemolysin-producing bacteria, large clear zones of hemolysis are observed that occurred after 4–6 h of incubation at 37?C (2 ). This chapter aims to describe three protocols useful for studying the hemolysin of enterohemorrhagic E. coli (EHEC): detection of the toxin, measurement of hemolytic activity, and, finally, determination of the pore-forming activity of the toxin. EHEC are a subgroup of Shiga toxin-producing E. coli (STEC, and are associated with severe human disease. It should be noted here that the enterohemolytic phenotype is not a characteristic property of all EHEC/STEC. Whereas almost all EHEC O157:H7 are enterohemolytic, the sorbitol-fermenting E. coli O157:H- are nonhemolytic. EHEC O111 differs in their capacity to produce E-Hly and EHEC O103:H2 overproduces E-Hly so that an α-hemolysin-like phenotype occurs on blood agar plates (3 ,4 ). In STEC, strains isolated from a variety of human and nonhuman sources also differ in their ability to produce EHEC-hemolysins.