Indole is produced by tryptophanase during growth of enteric bacteria and accumulates in the culture medium. The physiological role of indole production is poorly understood. We discovered that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 with a tnaA deletion has decreased secretion of EspA and EspB via the type III secretion system and as a result there is reduced formation of attaching and effacing (A/E) lesions in HeLa cells. Addition of indole restored and enhanced secretion of EspA and EspB and formation of A/E lesions by the tnaA deletion mutant EHEC. Indole addition moderately increased the promoter activity of LEE4 genes, including espA and espB, in the locus of enterocyte effacement. Thus in EHEC indole can serve to signal EspA and EspB expression and secretion and stimulate the ability of EHEC to form A/E lesions on human cells.