The H-NS protein is a major constituent of the Escherichia coli nucleoid structure and is implicated in the compact organization of the chromosome. Based on recent genetic evidence, this protein appears to influence the transcription of a variety of apparently unlinked genes on the chromosome, although the underlying molecular mechanism is not fully understood. In this study, we carried out a series of in vitro transcription assays including purified H-NS with special reference to the osmotically inducible proV promoter of the proVWX operon (or proU), whose expression is known to be derepressed by lesions of the hns (osmZ) gene. Here, H-NS was revealed to selectively inhibit an early step(s) of proV transcription initiation through its direct binding to the promoter region. It was thus demonstrated that H-NS functions directly as a transcriptional repressor. Under the in vitro conditions used, this in vitro inhibitory effect of H-NS was affected by changes in the superhelical density of template DNAs and more significantly by the concentration of potassium (K+) ions. These results are also discussed with regard to the mechanism underlying regulation of the proV promoter in response to the medium osmolarity.