A novel folding motif has been observed in four different proteins which bind oligonucleotides or oligosaccharides: staphylococcal nuclease, anticodon binding domain of asp-tRNA synthetase and B-subunits of heat-labile enterotoxin and verotoxin-1. The common fold of the four proteins, which we call the OB-fold, has a five-stranded beta-sheet coiled to form a closed beta-barrel. This barrel is capped by an alpha-helix located between the third and fourth strands. The barrel-helix frameworks can be superimposed with r.m.s. deviations of 1.4-2.2 A, but no similarities can be observed in the corresponding alignment of the four sequences. The nucleotide or sugar binding sites, known for three of the four proteins, are located in nearly the same position in each protein: on the side surface of the beta-barrel, where three loops come together. Here we describe the determinants of the OB-fold, based on an analysis of all four structures. These proposed determinants explain how very different sequences adopt the OB-fold. They also suggest a reinterpretation of the controversial structure of gene 5 ssDNA binding protein, which exhibits some topological and functional similarities with the OB-fold proteins.