H-NS is one of the two most abundant proteins in the bacterial nucleoid and influences the expression of a number of genes. We have studied the interaction of H-NS with DNA; purified H-NS was demonstrated to constrain negative DNA supercoils in vitro. This provides support for the hypothesis that H-NS influences transcription via changes in DNA topology, and is evidence of a structural role for H-NS in bacterial chromatin. The effects of H-NS on topology were only observed at sub-saturating concentrations of the protein. In addition, a preferred binding site on DNA was identified by DNase I footprinting at sub-saturating H-NS concentrations. This site corresponded to a curved sequence element which we previously showed, by in vivo studies, to be a site at which H-NS influences transcription of the proU operon. When present in saturating concentrations, H-NS did not constrain supercoils and bound to DNA in a sequence-independent fashion, covering all DNA molecules from end to end, suggesting that H-NS may form distinct complexes with DNA at different H-NS:DNA ratios. The data presented here provide direct support for the hypothesis that H-NS acts at specific sites to influence DNA topology and, hence, transcription.