The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively. We first isolated a number of hns mutations which resulted in derepression of the proVWX operon. These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS. The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS. This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties. Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA. The N-terminal domain was suggested to be involved in the ability to repress transcription. Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity. None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization. These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.