A small streptavidin-binding ligand was identified which incorporates some of the binding interactions seen in the crystal structures of streptavidin-biotin, and of streptavidin complexed with cyclo-Ac-[CHPQFC]-NH2, a cyclic peptide ligand with a K-d of 2.3 x 10(-7) M, discovered by phage display. The crystal structure of streptavidin-glycoluril is described and compared with the crystal structures of streptavidin-biotin and of streptavidin-cyclo-Ac-[CHPQFC]-NH2. The K-d of glycoluril for streptavidin was determined by plasmon resonance measurements to be 2.5 x 10(-6) M. The differences in the affinities of biotin and glycoluril for streptavidin were related to the differences in the crystal structures of the complexes and to differences in the determined solubilities of the ligands. Streptavidin-bound glycoluril has structural characteristics of, and makes interactions common to, both bound biotin and the bound cyclic peptide ligand. Binding of glycoluril and biotin is mediated by short, medium strength hydrogen bonds involving the ureido oxygen common to the two ligands. Binding of glycoluril is further mediated by a short hydrogen bond involving its unique ureido oxygen. The structural and physicochemical factors responsible for the weaker binding of glyeoluril compared with biotin an discussed.