The primary invasion factor of Yersinia enterocolitica, invasin, is encoded by inv. inv expression is regulated in response to pH, growth phase and temperature. In vitro, inv is maximally expressed at 26°C, pH 8.0, or 37°C, pH 5.5, in early stationary phase. At 37°C, pH 8.0, inv is weakly expressed. To identify which gene(s) are required for inv regulation, we screened for transposon insertions that decreased expression of an inv–′phoA chromosomal reporter at 26°C. Of 30 000 mutants screened, two were identified that had negligible inv expression in all conditions tested. Both of these independent mutants had an insertion into the same gene, designated rovA (regulator of virulence). RovA has 77% amino acid identity to the Salmonella typhimurium transcriptional regulator SlyA. Complementation with the wild-type rovA allele restores wild-type inv expression as monitored by Western blot analysis, tissue culture invasion assay and alkaline phosphatase assay. There is also a significant decrease in invasin levels in bacteria recovered from mice infected with the rovA mutant; therefore, RovA regulates inv expression in vivo as well as in vitro. In the mouse infection model, an inv mutant has a wild-type LD50, even though the kinetics of infection is changed. In contrast, the rovA mutant has altered kinetics, as well as a 70-fold increase in the LD50 compared with wild type. Furthermore, because the rovA mutant is attenuated in the mouse model, this suggests that RovA regulates other virulence factors in addition to inv. Analysis of other proposed virulence factors such as Ail, YadA and the Yop proteins shows no regulatory role for RovA. The more severe animal phenotype combined with the lack of impact on known virulence genes aside from inv suggests RovA regulates potentially novel virulence genes of Y. enterocolitica during infection.