The Escherichia coli DNA-binding protein H-NS is known to interact specifically with the upstream region of ribosomal RNA transcription units, where it causes transcriptional repression in vitro. Here, we present results demonstrating the effect of H-NS on rRNA transcription in vivo. rRNA synthesis rates were compared in cells that differ in the expression of functional H-NS or FIS molecules. We could show that in the absence of H-NS derepression of rRNA synthesis occurs at low growth rates. During the cell cycle H-NS is responsible for the rapid shut-off of rRNA synthesis at the end of the exponential phase. As it is known for FIS-dependent activation, the inhibitory function of H-NS is specific for P1, the first of the tandem rRNA promoters. The effect of H-NS on rRNA synthesis was further assessed under stress conditions. While under osmotic upshift the reduction in rRNA synthesis is clearly H-NS-dependent, no such influence could be detected at cold shock. Determination of the cellular ppGpp concentrations revealed that H-NS does not mediate its function via alterations in the synthesis of the global effector ppGpp.