Enteropathogenic Escherichia coli strains are able to signal host cells, cause dramatic cytoskeletal rearrangements, and adhere intimately to the cell surface in a process known as the attaching and effacing effect. A pathogenicity island of 35 kb known as the locus of enterocyte effacement (LEE) is necessary and sufficient for this effect. The LEE encodes an outer membrane adhesin called intimin, a type III secretion apparatus, and the EspA and EspB secreted proteins. The DNA sequence of the region between espA and espB revealed a new gene, espD. The product of espD was demonstrated by using a T7 expression system. We constructed a nonpolar mutation in espD and found that the mutant is incapable of the signal transduction events that lead to activation of the putative intimin receptor in host cells and that the mutant fails to induce the attaching and effacing effect. These phenotypes were restored to the mutant by complementation with a plasmid containing the cloned espD locus. We demonstrated by immunoblotting and microsequencing that the EspD protein is secreted via the type III apparatus. Thus, we describe a novel locus encoding a secreted protein that is required for attaching and effacing activity.