Upon infection of host cells, enteropathogenic Escherichia coli (EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn10kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including cesD, escC, escJ, escV, espD, sepL, espB, and escF. Other mutants contained insertions in micC or upstream of bfpP, yehL, or ydeP. The insertion upstream of ydeP was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes, ydeP and ydeO expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both ydeP and ydeO, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both ydeP and ydeO expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.