Enteropathogenic Escherichia coli (EPEC) expresses a type III secretion system (T3SS) required for pathogenesis. Regulation of the genes encoding the T3SS is complex; two major regulators control transcription, the silencer H-NS, and the related H-NS-like protein Ler. Our laboratory is interested in understanding the molecular differences that distinguish the anti-silencer Ler from H-NS, and how Ler differentially regulates EPEC virulence genes. Here, we demonstrate that mutated Ler proteins either containing H-NS alpha-helices 1 and 2, missing from Ler, or truncated for the 11 aa C-terminal extension compared with the related H-NS protein, did not appreciably alter Ler function. In contrast, mutating the proline at position 92 of Ler, in the conserved C-terminal DNA binding motif, eliminated Ler activity. Inserting 11 H-NS-specific amino acids, 11 alanines or 6 alanines into the Ler linker severely impaired the ability of Ler to increase LEE5 transcription. To extend our analysis, we constructed six chimeric proteins containing the N terminus, linker region or C terminus of Ler in different combinations with the complementary domains of H-NS, and monitored their in vivo activities. Replacing the Ler linker domain with that of H-NS, or replacing the Ler C-terminal, DNA binding domain with that of H-NS eliminated the ability of Ler to increase transcription at the LEE5 promoter. Thus, the linker and C-terminal domains of Ler and H-NS are not functionally equivalent. Conversely, replacing the H-NS linker region with that of Ler caused increased transcription at LEE5 in a strain deleted for hns. In summary, the interdomain linker specific to Ler is necessary for anti-silencing activity in EPEC.