The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic E. coli strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E. coli is associated with gain of this activity. Among 89 strains of E. coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.