Abstract The interaction of polypeptide (benzyloxycarbonyl derivatives of basic poly(γ-amino acid), benzyl esters of acidic poly(α-amino acid), and poly(l-leucine)) and lipid (fatty acids, fatty alcohols and cholesterol) at the air–water interface was investigated. From the additivity rule of mean molecular areas and the work of collapse of the monolayers, it was found that the characteristic interaction occurs in the polymer rich region (less than 0.2 lipid mol fraction), and that the interaction is stronger for the polymer with higher helical content. The interaction was found to take place between side chains of polypeptides and lipids, and to be mainly of hydrophobic nature. The components in mixed polypeptide–cholesterol monolayers exhibit characteristic miscibilities depending upon the chemical composition of polymer side chains. The polarized infrared spectra of collapsed films consisting of single or mixed components indicate that the polymer component is aligned in a direction parallel to the compressing barrier.