The isolated S-peptide (enzymatically cleaved 1-20 fragment of the ribonuclease, RNase S) is partly helical in solution. Both S-peptide and its truncated 1-15 analogue pep01 can combine with S-protein (residual part of RNase S without S-peptide), restoring a RNase S’ complex. Part 3-13 of the peptides forms an alpha-helix when within the RNase S’ complex. To compare the solvated forms of both peptides with their structures when complexed with the S-protein, we have measured the Raman spectra of these forms by means of difference spectroscopy. The spectra of the peptides in water solutions are characterized by much wider vibrational bands than the spectrum of the peptides within the RNase S’ complexes. This shows that the structure of the solvated peptides consists of a heterogeneous mixture of interconverting species. Only two main bands characteristic for the exposed alpha-helix (in D2O) and for an unordered chain are observed for amide-l regions of the dissolved peptides spectra. Relative intensities of the bands vary with temperature, reflecting different proportions of helical and disordered conformations. However, amide-I bands compositions of the bound peptides are more complex and include marker bands typical of the alpha-helix within the hydrophobic environment of a protein.