The transcriptional activator RovA of Yersinia pseudotuberculosis, a member of the SlyA/Hor family, activates its own expression and that of the virulence factor invasin in response to moderate growth temperature, but not at 37 degrees C. In this work, we analysed the mechanism of RovA-dependent transcription of the rovA and inv genes. We found that rovA is transcribed by two different promoters. Sequences located upstream and downstream of the promoters were involved in rovA autoregulation and interacted specifically with the RovA protein. To define the nucleotides recognized by the RovA protein, we determined the RovA binding sites in the rovA and the inv regulatory region and revealed related AT-rich sequence motifs at diverse positions relative to the transcriptional start sites. We also showed that rovA and the RovA-dependent inv gene were both subject to silencing by the nucleoid-associated H-NS protein of Y. pseudotuberculosis. The binding sites of the H-NS and RovA proteins in the rovA and inv regulatory sequences were superimposed, and the presence of the RovA protein alleviated H-NS-mediated repression of the rovA and inv promoter. Moreover, loss of H-NS function led to a significant increase in rovA and inv transcription nearly independently of RovA, indicating that RovA acts mainly as an antirepressor. We therefore hypothesize that the transcription level of RovA-dependent genes reflects the outcome of the RovA/H-NS competition and the rovA autoregulatory mechanism.