The locus of enterocyte effacement (LEE) pathogenicity island is required for the intimate adhesion of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelial cells. GrlR and GrlA are LEE-encoded negative and positive regulators, respectively. The interaction of these two regulators is important for controlling the transcription of LEE genes through Ler, a LEE-encoded central activator for the LEE. The GrlR-GrlA regulatory system controls not only LEE but also the expression of the flagellar and enterohemolysin (Ehx) genes in EHEC. Since Ehx levels were markedly induced in a grlR mutant but not in a grlR grlA double mutant and significantly increased by overexpression of GrlA in a ler mutant, GrlA is responsible for this regulation (T. Saitoh et al., J. Bacteriol. 190:4822-4830, 2008). In this study, additional investigations of the regulation of ehx gene expression determined that Ler also acts as an activator for Ehx expression without requiring GrlA function. We recently reported that the LysR-type regulator LrhA positively controls LEE expression (N. Honda et al., Mol. Microbiol. 74:1393-1411, 2009). The hemolytic activity of the lrhA mutant strain of EHEC was lower than that of the wild-type strain, and LrhA markedly induced ehx transcription in an E. coli K-12 strain, suggesting that LrhA also activates the transcription of ehx without GrlA and Ler. Gel mobility shift assays demonstrated that Ler and LrhA directly bind to the regulatory region of ehxC. Together, these results indicate that transcription of ehx is positively regulated by Ler, GrlA, and LrhA, which all act as positive regulators for LEE expression.