In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3 mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5 C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3 mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation time, as well as the fluorescence quenching of DPH by TEMPO, are at maxima, while the fluorescence intensity of dehydroergosterol and the lipid solubility in TritonX-100 are at minima. These results correlate well with the hypothesis of domain segregation in the DMPC/DSPC/Cholesterol LUV system. In this context, we postulate that at 33.3 mol% of Cho, the proportion of ordered domains reaches a maximum.