Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at -313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild-type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid-log cultures and negligible in cells entering the stationary phase. The level of repression in early-log phase was lower than in mid-log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H-NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H-NS and Fis protein to transcriptional systems programmed with the same hns-cat fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation gel analyses revealed that H-NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around -150. When these sites are filled by H-NS, an additional site between approximately -20 and -65, which partly overlaps the promoter, is also occupied. Binding of H-NS to this site is probably the ultimate cause of transcriptional auto-repression.