Peptides identified by the PD method against a film surface composed of azobenzene-containing synthetic polymers were characterized by QCM measurements. Among the four peptides analyzed, the c16 peptide with the sequence Trp-His-Thr-Leu-Pro-Asn-Ala showed the highest binding affinity to the films rich in cis-azobenzene groups. SPR served to determine the association/dissociation constants of the c16 peptide against the trans- and cis-azobenzene groups. The binding constants were estimated to be 1.3 × 105 and 1.4 × 106/M, respectively, indicating a high specificity of the c16 peptide for cis-azobenzene conformer. Then mutants of the c16 peptide were synthesized and characterized to gain information on the structural requirements for the cis-form specificity. An Ala-scan clearly revealed that all the amino acid residues of the c16 peptide were essential for the specificity. More importantly, the results with peptides of inverted sequence and containing Gly insertions confirmed that the specificity is strictly derived from the primary sequence of the c16 peptide. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.