During infections of the intestinal tract Yersinia pseudotuberculosis penetrates the epithelial cell layer through M-cells into the Peyer's patches. This early step in the infection process is primarily mediated by the outer membrane protein invasin. Expression of the invasin gene is activated by the MarR-type regulatory protein RovA in response to environmental conditions, including temperature and growth phase. In order to gain insight into the nature of the underlying control systems, mutagenesis and gene bank screens were used to identify regula components modulating the levels of invasin and RovA. We found that the inv and rovA genes were both subjected to silencing by the nucleoid-associated protein H-NS. Under inducing conditions, RovA appears to disrupt the silencer complex, through displacement of H-NS from an extended AT-rich region located upstream of the inv and rovA promoters. Furthermore, a LysR-type regulatory protein, RovM with homology to HexA/PecT of phytopathogenic Erwinia species was shown to interact specifically with the rovA regulatory region and represses rovA transcription in addition to H-NS. Disruption of the rovM gene significantly enhanced internalization of Y. pseudotuberculosis into host cells and higher numbers of the mutant bacteria were detectable in gut-associated lymphatic tissues and organs in infected mice. In addition, the histone-like protein YmoA, which has a global effect on the bacterial physiology, was found to activate rovA expression through RovM. Together, our studies showed, that H-NS, RovM and YmoA are key regulators implicated in the environmental control of virulence factors, which are important for the initiation of a Yersinia infection.