A multidisciplinary panel of leading experimentalists details the key protocols for the diagnosis and study of the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) and its isolated cytotoxin (Stx). Described in step-by-step detail, these readily reproducible techniques range from those for the diagnosis and detection of STEC bacteria in patient and animal samples, to those for studying the cellular microbiology of STEC infections, especially host–pathogen interactions and the hemolysin of STEC. There are also protocols for studying the details of Shiga toxin (Stx) biology—from the purification of the toxin to the effects of Stx on various host cell functions—and for exploring STEC-mediated disease in various animal models.