Liposomes composed of cationic lipids and neutral phospholipids have been used as vehicles for cellular delivery of DNA and drug delivery. The aim of this study is to get a better understanding of phospholipid–cationic lipid interactions that is essential for the development of this potential. We have studied the interaction between cationic lipid liposomes primarily composed of dioctadecyldimethylammonium bromide (DODAB) with two different phospholipids, dioleoyl phosphatidylethanolamine (DOPE) and soybean phosphatidylcholine (PC), as well as with cholesterol. The effect of these components on the liposome’s physical properties (microviscosity and polarity) was accessed by studying, both the steady-state and time-resolved, fluorescence anisotropy of the dye Nile Red. This information combined with analysis of the steady-state emission and fluorescence lifetime of Nile Red using the different lipids and cholesterol provided information concerning the polarity and hydration level changes in these vesicle systems along with organisational information.