EPR spin-labeling methods were used to investigate the order and fluidity of alkyl chains, the hydrophobicity of the membrane interior, and the order and motion of cholesterol molecules in coexisting phases and domains, or in a single phase of fluid-phase cholesterol/egg-sphingomyelin (Chol/ESM) membranes with a Chol/ESM mixing ratio from 0 to 3. A complete set of profiles for these properties was obtained for the liquid-disordered (l (d)) phase without cholesterol, for the liquid-ordered (l (o)) phase for the entire region of cholesterol solubility in this phase (from 33 to 66 mol%), and for the l (o)-phase domain that coexists with the cholesterol bilayer domain (CBD). Alkyl chains in the l (o) phase are more ordered than in the l (d) pure ESM membrane. However, fluidity in the membrane center is greater. Also, the profile of hydrophobicity changed from a bell to a rectangular shape. These differences are enhanced when the cholesterol content of the l (o) phase is increased from 33 to 66 mol%, with clear brake-points between the C9 and C10 positions (approximately where the steroid-ring structure of cholesterol reaches into the membrane). The organization and motion of cholesterol molecules in the CBD are similar to those in the l (o)-phase domain that coexists with the CBD.