It is well-known that the CdropN stretching vibration in acetonitrile is sensitive to solvent. Therefore, we proposed in this contribution to use this vibrational mode to report local environment of a particular amino acid in proteins or local environmental changes upon binding or folding. We have studied the solvent-induced frequency shift of two nitrile-derivatized amino acids, which are, Ala(CN) and Phe(CN), in H2O and tetrahydrofuran (THF), respectively. Here, THF was used to approximate a protein's hydrophobic interior because of its low dielectric constant. As expected, the CdropN stretching vibrations of both Ala(CN) and Phe(CN) shift as much as similar to10 cm(-1) toward higher frequency when THF was replaced with H2O, indicative of the sensitivity of this vibration to solvation. To further test the utility of nitrile-derivatized amino acids as probes of the environment within a peptide, we have studied the binding between calmodulin (CaM) and a peptide from the CaM binding domain of skeletal muscle myosin light chain kinase (MLCK579-595), which contains a single Phe(CN). MLCK579-595 binds to CaM in a helical conformation. When the Phe(CN) was substituted on the polar side of the helix, which was partially exposed to water, the CdropN stretching vibration is similar to that of Phe(CN) in water. In constrast, when Phe(CN) is introduced at a site that becomes buried in the interior of the protein, the CdropN stretch is similar to that of Phe(CN) in THF. Together, these results suggest that the CdropN stretching vibration of nitrile-derivatized amino acids can indeed be used as local internal environmental markers, especially for protein conformational studies.