The separation of dipeptide and tripeptide enantiomers using a neutral single isomer cyclodextrin (CD) derivative, heptakis-(2,3-di-O-acetyl)-beta-CD (DIAC-beta-CD), was investigated with respect to the amino acid sequence applying standard separation conditions. With only one exception the DD-enantiomers migrated faster than the LL-stereoisomers. Separations obtained for the same set of peptides using beta-CD and the sulfated single isomer derivatives heptakis-(2,3-di-O-acetyl-6-sulfo)-beta-CD (HDAS-beta-CD) and heptakis-6-sulfo-beta-CD (HS-beta-CD) revealed identical enantiomer migration order in the presence of the 2,3-disubstituted derivatives DIAC-beta-CD and HDAS-beta-CD. In contrast, reversed migration sequence was found for beta-CD and HS-beta-CD compared to DIAC-betaCD and HDAS-beta-CD indicating the importance of the substitution pattern on the wider rim of the CD cavity on the chiral recognition of the peptide enantiomers by the CDs. Nuclear magnetic resonance (NMR) experiments indicated different complexation modes between the enantiomers and the CDs depending on the presence of acetyl substituents on the wider rim of the CD torus. Thus, the CD-induced chemical shifts observed in samples containing Ala-Phe or Ala-Tyr and beta-CD or HS-beta-CD were consistent with an inclusion of the aromatic moiety into the CD cavity. Although the CD-induced chemical shifts in the presence of DIAC-beta-CD and HDAS-beta-CD did not allow direct conclusions on the complexation mode they substantially differed from those observed in the presence of 2,3-unsubstituted CDs indicating different structures of the peptide-CD complexes.