High affinity cyclic thioether-cross-linked streptavidin binding ligands were designed from the crystal structure of a complex between streptavidin and a high affinity cyclic disulfide-cross-linked peptide ligand, cyclo-Ac-[CHPQGPPC]-NH2, originally discovered by phage display. Determination of the affinities of two chemically synthesized thioether ligands by surface plasmon resonance indicated affinities similar to the disulfide-cross-linked ligand from which they were designed. The crystal structures of the streptavidin-cyclic thioether complexes show that the nonlinker segments are bound in the same conformation as in cyclo-Ac-[CHPQGPPC]-NH2 and make the same binding interactions with streptavidin as the disulfide-linked cyclic peptide. The structures, conformations, and dihedral energetic constraints of the thioether- and disulfide-cross-linked ligands are described and compared. Advantages of thioether cross-links over disulfide cross-links are discussed.