Fine effects of the hydration, charge, and conformational structural changes in L-alanyl-L-alanine (Ala-Ala) dipeptide were studied with the aid of Raman and Raman optical activity (ROA) spectra. The spectra were recorded experimentally and analyzed by means of density functional computations. A (15)N and (13)C isotopically labeled analogue was synthesized and used to verify the vibrational mode assignment. Calculated shifts in vibrational frequencies for isotopically labeled molecule agreed well with the experiment. The assignment made it possible to scale computed vibrational frequencies and extract better structural information from the intensities. Solvent modeling with clusters obtained from molecular dynamics led to a qualitatively correct inhomogeneous broadening of Raman spectral lines but did not bring a convincing improvement of ROA signal when compared to a standard dielectric solvent correction. In comparison with the zwitterionic form, charged anionic and cationic dipeptides provided spectral variations that indicated different conformational behavior. Only minor backbone conformational change occurs in the cation, whereas the results indicate the presence of more anion conformers differing in the rotation of the NH(2) group and the backbone psi-angle. These findings are in agreement with previous electronic circular dichroism (ECD) and NMR studies. The results confirm the large potential of the ROA technique for the determination of final details in molecular structure and conformation.