We report unambiguous spectroscopic evidence of peptide bond formation at the air-water interface, yielding a possible mechanism providing insight into the formation of modern ribosomal peptide bonds, and a means for the emergence of peptides on early Earth. Protein synthesis in aqueous environments, facilitated by sequential amino acid condensation forming peptides, is a ubiquitous process in modern biology, and a fundamental reaction necessary in prebiotic chemistry. Such reactions, however, are condensation reactions, requiring the elimination of a water molecule for every peptide bond formed, and are thus unfavorable in aqueous environments both from a thermodynamic and kinetic point of view. We use the hydrophobic environment of the air-water interface as a favorable venue for peptide bond synthesis, and demonstrate the occurrence of this chemistry with in situ techniques using Langmuir-trough methods and infrared reflection absorption spectroscopy. Leucine ethyl ester (a small amino acid ester) first partitions to the water surface, then coordinates with Cu 2þ ions at the interface, and subsequently undergoes a condensation reaction selectively forming peptide bonds at the air-water interface.