Ascorbyl palmitate formed vesicles in combination with cholesterol and a negatively charged lipid (dicetyl phosphate). Niosomes were prepared by the film hydration method, followed by sonication, in which aqueous doxorubicin solution (in phosphate buffer saline pH=7.4) was encapsulated in aqueous regions of lipid layers. Vesicle's formation was confirmed by either scanning electron microscopy images or observation of unsonicated vesicles by light microscope. The percent efficiencies of doxorubicin encapsulation of the different formulations were determined by fluorescence spectrophotometry. Cholestrol content in niosomes did not exhibit any relation to vesicle size, zeta potential, percent entrapment, or release rate. Differential scanning calorimetric data of pure lipids, vesicle dispersion, and mixture of lipids confirmed the formation of niosomes.