An artificial 24-residue DDT-binding polypeptide (Moser, R., Thomas, R.M., and Gutte, B. (1983) FEBS Lett. 157, 247-251) and several analogues of this peptide were characterized by ligand binding, spectroscopic, and immunological studies. Comparison of dissociation constants showed that Phe14 and His16 were important for DDT binding and that the designed peptide had noticeable ligand specificity. Measurement of the circular dichroism of the artificial DDT-binding peptide revealed a high proportion of beta-structure which was abolished only partly by 8 M urea. When Tyr15, Tyr17, and Phe3 whose side chains were on the same side of the proposed beta-sheet were replaced by non-aromatic amino acids, the cross-reactivity with antibodies against the original DDT-binding peptide decreased stepwise. In summary, the results of this study support essential features of our structural model of the designed 24-residue DDT-binding peptide.