The human, pig, and frog neurotensins and four single-site mutants of human neurotensin (NT), having the following modifications, [Gln(4)]NT, [Trp(11)]NT, [D-Trp(11)]NT, and [D-Tyr(11)]NT, were immobilized onto an electrochemically roughened silver electrode surface in an aqueous solution. The orientation of adsorbed molecules was determined from surface-enhanced Raman scattering (SERS) measurements. A comparison was made between these structures to determine how the change upon the mutation of the neurotensin structure influences its adsorption properties. The SEAS patterns were correlated with the contribution of the structural components of the aforementioned peptides to the ability to interact with the NTR1 G-protein receptor. Briefly, the SERS spectra revealed that the substitution of native amino acids in investigated peptides influenced slightly their adsorption state on an electrochemically roughened silver surface. Thus, human, pig, and frog neurotensins and [Gln(4)]NT and [D-Tyr(11)]NT tended to adsorb to the surface via the tyrosine ring, the oxygen atom of the deprotonated phenol group of Tyr(11), and the -CH(2)- unit(s), most probably of Tyr(11), Arg(9), and/or Leu(13). The observed changes in the enhancement of the deprotonated Tyr residue SERS signals indicated a further parallel orientation of a phenol-O bond with regard to the silver surface normal for pig NT, [Cln(4)]NT, and [D-Tyr(11)]NT, whereas the orientation was slightly tilted for human and frog NT. In the case of [Trp(11)]NT and [D-Trp(11)]NT, the formation of a peptide/Ag complex was confirmed by strong SERS bands involving the phenyl co-ring of Trp(11)/D-Trp(11) and -CH(2)- vibrations and the tilted and flat orientations of the two compounds with respect to the surface substrate. The spectral features were accompanied by a SERS signal caused by vibrations of the carboxyl group of C-terminal Leu(13) and the guanidine group of Arg(9). Reported changes in SERS spectra of L and D isomers were fully supported by generalized two-dimensional correlation analysis. Additionally, a combination of mutation-labeling and vibrational spectroscopy (Fourier-transform Raman and absorption infrared) was used to investigate the possible peptide conformations and environments of the tyrosine residues.