The DNA-binding domain of the single-stranded DNA-binding protein IKe GVP was studied by means of 1H nuclear magnetic resonance, through use of oligonucleotides of two and three adenyl residues in length, that were spin-labelled at their 3′ and/or 5′ termini. These spin-labelled ligands were found to cause line broadening of specific protein resonances when bound to the protein, although they were present in small quantities, i.e. of the order of 0.04 molar equivalent and less. The line broadening of protein resonances was made manifest by means of difference one and two-dimensional spectroscopy. Difference one-dimensional experiments revealed line broadening of the same protein resonances upon binding of either 3′ or 5′ spin-labelled oligonucleotides. Evidence in favour of the existence of a fixed 5′ to 3′ orientation in the binding of oligonucleotides to the protein surface was therefore not obtained from the spin-labelled oligonucleotide binding studies. Residue-specific assignments of broadened resonances could not, or could only sparsely, be derived from the difference one-dimensional spectra, because of the tremendous overlap in the aliphatic region of the spectrum. In contrast, such assignments were easily obtainable from the difference two-dimensional spectra, which were recorded by means of both total correlated spectroscopy and nuclear Overhauser effect spectroscopy. Difference signals were detected for 15 spin systems; ten out of these were assigned to the residues I29, Y27, S20, G18, R16, T28, K22, Q21, V19 and S17 in the amino acid sequence of IKe GVP; the other five spin systems could be assigned to a phenylalanyl residue, an arginyl or lysyl residue, an aspartic acid or asparagyl residue, a glycyl residue and a glutamic acid or glutamyl residue. From the evaluation of the relative difference signals, it was concluded that the direct surroundings of the spin-label group of the labelled oligonucleotide in the bound state is composed of the first five residues in the former group of residues and the five residues in the latter group. Further analysis of the relative difference signals detected for the residues that are part of the DNA-binding wing of IKe GVP, i.e. the amino acid sequence segment running from R16 to I29, leads to the conclusion that the spin-labelled oligonucleotide is in the bound state in close contact with the surface formed by the side-chains of residues I29, Y27, S20, G18 and R16, which constitute one side of the β-ladder structure of the DNA binding wing, and that the other side of this β-ladder structure, formed by the side-chains of residues S17, V19, K25, P26 and T28, does not form part of the oligonucleotide-binding site. The binding-site model for IKe GVP, as put forward on the basis of the oligonucleotide-binding studies, is strongly supported by the existence of a unique sequence identity among different single-stranded DNA-binding proteins. It is postulated that the mechanisms by which the DNA-binding wings of the single-stranded DNA-binding proteins of the filamentous phages IKe GVP, Ff GVP and Pf3 DBP interact with single-stranded DNA are highly similar. Moreover, the sequence identity among these proteins and that among IKe GVP, the single-stranded DNA-binding protein G32P of bacteriophage T4 and the protein RecA of Escherichia coli, suggest that a certain class of single-stranded DNA-binding proteins utilizes, as part of their DNA-binding domain, a β-loop with conserved amino acid residues to bind single-stranded DNA.