Enterohemorrhagic Escherichia coli (EHEC) is a prominent, food-borne cause of diarrhea, bloody diarrhea, and the hemolytic uremic syndrome in industrialized countries. Most strains of EHEC carry the locus for enterocyte effacement (LEE) pathogenicity island, but a proportion of isolates from patients with severe disease do not carry LEE and very little is known about virulence factors in these organisms. LEE-negative strains of EHEC typically express Shiga toxin 2 and carry a large plasmid that encodes the production of EHEC hemolysin. In this study, we determined the nucleotide sequence of the transfer region of pO113, the large hemolysin plasmid from LEE-negative EHEC O113:H21 (EH41). This 63.9-kb region showed a high degree of similarity with the transfer region of R64, and pO113 was capable of self-transmission at low frequencies. Unlike R64 and the related dot/icm system of Legionella pneumophila, however, pO113 was unable to mobilize RSF1010. In addition, the pO113 transfer region encoded a novel high-molecular-weight serine protease autotransporter of Enterobacteriaceae (SPATE) protein, termed EpeA. Like other SPATEs, EpeA exhibited protease activity and mucinase activity, but expression was not associated with a cytopathic effect on epithelial cells. Analysis of a second high-molecular-weight secreted protein revealed that pO113 also encodes EspP, a cytopathic SPATE identified previously in EHEC O157:H7. The nucleotide sequences encoding the predicted beta-domains of espP and epeA were identical and also shared significant homology with a third SPATE protein, EspI. Both espP and epeA were detected in several LEE-negative clinical isolates of EHEC and thus may contribute to the pathogenesis of this subset of EHEC.