AbstractSolid‐state protonated and N,O‐deuterated Fourier transform infrared (IR) and Raman scattering spectra together with the protonated and deuterated Raman spectra in aqueous solution of the cyclic di‐amino acid peptide cyclo(L‐Asp‐L‐Asp) are reported. Vibrational band assignments have been made on the basis of comparisons with previously cited literature values for diketopiperazine (DKP) derivatives and normal coordinate analyses for both the protonated and deuterated species based upon DFT calculations at the B3‐LYP/cc‐pVDZ level of the isolated molecule in the gas phase. The calculated minimum energy structure for cyclo(L‐Asp‐L‐Asp), assuming C2 symmetry, predicts a boat conformation for the DKP ring with both the two L‐aspartyl side chains being folded slightly above the ring. The CO stretching vibrations have been assigned for the side‐chain carboxylic acid group (e.g. at 1693 and 1670 cm−1 in the Raman spectrum) and the cis amide I bands (e.g. at 1660 cm−1 in the Raman spectrum). The presence of two bands for the carboxylic acid CO stretching modes in the solid‐state Raman spectrum can be accounted for by factor group splitting of the two nonequivalent molecules in a crystallographic unit cell. The cis amide II band is observed at 1489 cm−1 in the solid‐state Raman spectrum, which is in agreement with results for cyclic di‐amino acid peptide molecules examined previously in the solid state, where the DKP ring adopts a boat conformation. Additionally, it also appears that as the molecular mass of the substituent on the Cα atom is increased, the amide II band wavenumber decreases to below 1500 cm−1; this may be a consequence of increased strain on the DKP ring. The cis amide II Raman band is characterized by its relatively small deuterium shift (29 cm−1), which indicates that this band has a smaller NH bending contribution than the trans amide II vibrational band observed for linear peptides. Copyright © 2009 John Wiley & Sons, Ltd.