Vibrational couplings between the amide modes are keenly dependent on peptide structure. Site-specific couplings can inform us of molecular conformation in detail. For example, when an amide-I mode couples to an amide-II mode that is three residues away because they are brought into proximity in the presence of an intramolecular C=O...H-N hydrogen bond, the coupling can provide direct evidence for single helical turn formation, a proposed key step in coil-helix transition. In this work, we measure 2D IR spectra of a 3(10)-helical hexapeptide, Z-Aib-l-Leu-(Aib)(2)-Gly-Aib-OtBu, and its (13)C=(18)O-Leu monolabeled and (13)C=(18)O-Leu/(15)N-Gly bis-labeled isotopomers in CDCl(3). The isotope-dependent amide-I/II cross-peaks clearly reveal the existence of vibrational coupling between the second and fourth peptide linkages that are connected through a 3(10)-helical hydrogen bond. Our results demonstrate that the combination of 2D IR and (13)C=(18)O/(15)N labeling is a useful structural method for probing local peptide conformation with residue-level specificity.