The thermodynamic binding parameters and crystal structure for streptavidin-peptide complexes where the peptide sequences were obtained by random screening methods are reported. The affinities between streptavidin and two heptapeptides were determined by titrating calorimetric methods [Phe-Ser-His-Pro-Gln-Asn-Thr, Ka = 7944 (+/- 224) M-1, delta G degrees = -5.32 (+/- 0.01) kcal/mol, and delta H degrees = -19.34 (+/- 0.48) kcal/mol; His-Asp-His-Pro-Gln-Asn-Leu, Ka = 3542 (+/- 146) M-1, delta G degrees = -4.84 (+/- 0.03) kcal/mol, and delta H degrees = -19.00 (+/- 0.64) kcal/mol]. The crystal structure of streptavidin complexed with one of these peptides has been determined at 2.0-A resolution. The peptide (Phe-Ser-His-Pro-Gln-Asn-Thr) binds in a turn conformation with the histidine, proline, and glutamine side chains oriented inward at the biotin-binding site. A water molecule is immobilized between the histidine and glutamine side chains of the peptide and an aspartic acid side chain of the protein. Although some of the residues that participate in binding biotin also interact with the screened peptide, the peptide adopts an alternate method of utilizing binding determinants in the biotin-binding site of streptavidin.