In our recent studies, the reversal of the enantiomer migration order (EMO) was observed with heptakis (2,3-dimethyl-6-sulfo)-beta-CD (HDMS-beta-CD) when aqueous electrolyte was changed with nonaqueous electrolyte in CE. One-dimensional rotating frame nuclear Overhauser effect spectroscopy experiments prevailed that an inclusion complex was formed between the analyte and the chiral selector in the aqueous buffer, whereas an external complex resulted when a methanolic electrolyte was employed. In the case of the similarly substituted heptakis (2,3-diacetyl-6-sulfo)-beta-CD (HDAS-beta-CD), the external complex was observed in the aqueous buffer but an inclusion complex was formed in methanolic electrolyte. In contrast to heptakis (2,3-dimethyl-6-sulfo)-beta-CD, no reversal of the enantiomer migration order was observed with HDAS-beta-CD. In the present study, further mechanisms of enantioselective recognition and separation of propranolol enantiomers with HDAS-beta-CD were investigated by using different techniques of nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. To the best of our knowledge, enantioselective nuclear Overhauser effect was observed for the first time in this study.