Abstract Peptide display on solvent-exposed surfaces of carrier proteins is a promising approach pursuing the identification and improvement of reactive amino acid sequences. However, the contribution of the molecular environment where the peptide is inserted on its interactive properties remains essentially unexplored. By an exhaustive antigenic analysis of the same peptide displayed on 20 structurally distinct frameworks, we show that peptide accommodation into the acceptor site has dramatic effects on its immunoreactivity. Conformational constraints can modulate the molecular recognition properties of the insert within a surprisingly wide range, probably by affecting the positioning of critical contact residues. The observed display-induced antigenic variation prompts a careful consideration of the molecular context when evaluating output amino acid sequences from screening of peptide libraries or application of directed molecular evolution technologies.