H-NS is an abundant structural component of bacterial chromatin and influences many cellular processes, including recombination, transposition, and transcription. We have studied the mechanism of action of H-NS at the osmotically regulated proU promoter. The interaction of H-NS with a curved DNA element located downstream of the proU promoter is required for normal regulation of expression. Heterologous curved sequences can replace the regulatory role of the proU curve. Hence, the luxAB and lacZ reporter genes, which differ in the presence or absence of a curve, can indicate very different patterns of transcription. H-NS interacts preferentially with these curved DNA elements in vitro. Furthermore, in vivo the interaction of H-NS with curved DNA participates in the control of plasmid linking number. The data suggest that H-NS-dependent changes in DNA topology play a role in the osmoregulation of proU expression.