Carbonyl–carbonyl interactions between adjacent backbone amides have been implicated in the conformational stability of proteins. By combining experimental and computational approaches, we show that relevant amidic carbonyl groups associate through an n→π* donor–acceptor interaction with an energy of at least 0.27 kcal/mol. The n→π* interaction between two thioamides is 3-fold stronger than between two oxoamides due to increased overlap and reduced energy difference between the donor and acceptor orbitals. This result suggests that backbone thioamide incorporation could stabilize protein structures. Finally, we demonstrate that intimate carbonyl interactions are described more completely as donor–acceptor orbital interactions rather than dipole–dipole interactions.