The role of the hns gene (coding for the curved-DNA-binding protein H-NS) in the thermo-osmotic regulation of Shigella flexneri virulence gene transcription was investigated. Two structural genes, mxiC and icsB, which are transcribed divergently on the high-molecular-weight virulence plasmid, were found to be transcriptionally inhibited in cultures grown in a low-osmolarity medium, even at the inducing temperature. This repression was relieved by inactivation of the hns gene, establishing a role for hns in the osmotic as well as the thermal regulation of invasion gene expression. The physiological relevance of this finding is discussed.