The interaction between nucleic acids and Escherichia coli H-NS, an abundant 15 kDa histone-like protein, has been studied by affinity chromatography, nitrocellulose filtration and fluorescence spectroscopy. Intrinsic fluorescence studies showed that the single Trp residue of H-NS (position 108) has a restricted mobility and is located within an hydrophobic region inaccessible to both anionic and cationic quenchers. Binding of H-NS to nucleic acids, however, results in a change of the microenvironment of the Trp residue and fluorescence quenching; from the titration curves obtained with addition of increasing amounts of poly(dA)-poly(dT) and poly(dC)-poly(dG) it can be estimated that an H-NS dimer in 1.5 x SSC binds DNA with an apparent Ka approximately equal to 1.1 x 10(4) M-1.bp-1. H-NS binds to double-stranded DNA with a higher affinity than the more abundant histone-like protein NS(HU) and, unlike NS, prefers double-stranded to single-stranded DNA and DNA to RNA; both monovalent and divalent cations are required for optimal binding.