The disposition of cholesterol inside the β-cyclodextrin cavity (β-CD) was deduced from oxidation of cholesterol secondary alcohol groups by Ca(OCl)2 and H2O2 in the pyridine–acetic acid system. The amount of cholest-4-ene-3-one formed was found to be proportional to the concentration of β-cyclodextrin, resulting in 56.1% of ketone. The oxidation rate was enhanced by β-cyclodextrin and its methyl, polymer and 1:1 copper(II)–β-cyclodextrin derivatives. Detailed investigations involving UV-visible, 13C- and 1H-NMR (T1, 1D NOE and ROESY) spectroscopic studies were carried out. A binding constant value of 15,385 ± 1500 M-2 was obtained for the 2:1 heptakis-2,6-di-O-methyl-β-cyclodextrin(DMβ-CD): cholesterol complex in chloroform from UV studies. Proton and solid state13C-CP MAS spectra of the β-CD–cholesterol mixture showed large magnitude shifts for the protons from the wider end of the β-CD cavity as well as those of ring A and ring B of cholesterol. Both 1D NOE and ROESY measurements indicated the proximity between ring A and ring B protons of cholesterol and the wider end protons of β-CD and DMβ-CD. Besides, analysis of τc, τi and tau;m from T1 measurements showed not only a lowering of rotational motions but a χ value of 0.016–0.048 for some of the cholesterol protons, typical of a weak complex. Based on these studies, a probable structure for the 2:1 complex involving two molecules of β-CD/DMβ-CD was proposed with portions of ring A and ring B being present inside the wider end of the β-CD/DMβ-CD cavity and ring D and the side chain attached at position 17, projecting into the wider end of the second βCD/DMβ-CD molecule.