Background: Gastroenteritis caused by Salmonella Typhimurium is triggered by bacterial adherence to intestinal epithelium cells that leads to invasion and destruction of the mucosal surface. The pef operon of S. Typhimurium is responsible for the biosynthesis of plasmid-encoded fimbriae (Pef), which mediate adhesion to mouse intestinal epithelium. As most of the 13 fimbriae of S. Typhimurium, expression of Pef fimbriae is tightly regulated. In vitro, their expression was previously detected only in standing cultures grown in rich acidic medium. Previous work and microarray studies suggested a role of the nucleoproteins H-NS and Hha-YdgT in this negative regulation of pef fimbriae expression. In this study, our objective was to demonstrate this repression and to characterize the underlying mechanism. Methods: Due to instability of hns mutants, strains carrying a deletion of the hns gene were freshly constructed by P22 transduction before each experiment. Promoters activities were quantified using plasmid-based transcriptional fusions carried by wild-type, hns and/or hha-ydgT mutants. Expression of Pef fimbriae was measured by RT-PCR and Western blot by measuring pefA/PefA expression, which encodes the major subunit. Results: We demonstrate that H-NS and Hha-YdgT negatively regulate pef operon transcription by acting on the promoter located upstream of pefB, the first gene of the operon. The effect of H-NS was much more pronounced than that of Hha-YdgT. Moreover, we observed that Hha and YdgT can repress pef expression independently of H-NS when bacteria were cultivated in acidic medium under standing conditions, but not after culture in neutral pH medium. Conclusions: This work demonstrates that the weak expression of Pef fimbriae in vitro is partly due to the combined action of H-NS and Hha-YdgT nucleoproteins on the transcriptional activity of the promoter region located upstream of the pef operon. A debate still exists in the literature concerning the exact mode of action of these nucleoproteins. Experimental evidence and a mechanistic model recently described indicate that Hha and YdgT act primarily through H-NS to modulate gene expression. On the contrary, few reports show that Hha can bind to specific regulatory sequences independently of H-NS. Our results on pef operon transcriptional regulation are in favor of the existence of these two models. Indeed, Hha and YdgT can act through H-NS to modulate pef expression. Nevertheless, according to the culture conditions used in our experiments, it appears that Hha and YdgT can also act independently of H-NS.