The linear infrared and two-dimensional infrared (2D IR) spectra in the amide-I region of N-acetyl tryptophan methyl amide (NATMA) in solvents of varying polarity are reported. The two amide-I transitions have been assigned unambiguously by using 13C isotopic substitution of the carbonyl group. The amide unit at the amino end shows a lower transition frequency in CH2Cl2 and methanol, while the acetyl end has a lower transition frequency in D2O. Multiple conformers exist in CH2Cl2 and methanol, but only one conformer is evident in D2O. The 2D IR cross peaks from the intermode coupling yield off-diagonal anharmonicities, which by simple matrix diagonalization yield the coupling constants (8.0�0.5 cm-1), (8.0�1.0 cm-1) and (5.5�1.0 cm-1) in CH2Cl2, methanol and D2O respectively. The major conformer in CH2Cl2 corresponds to a C7 structure, in agreement with that found in the gas phase with intramolecular hydrogen bonding between the acetyl end C=O and the amino end N-H. The backbone dihedral angles (�, �) are determined for NATMA in CH2Cl2, methanol and D2O respectively. This work was supported by NIH (RR 01348 and GM12592) and by NSF CHE.