The 3-D morphology of doxorubicin (DOX)-loaded liposomes with a size of ca. 100 nm was characterized by AFM in an aqueous environment. Prolate liposomes appear in accordance with linear expansion of DOX fiber bundles precipitated inside liposomes. Oblate and concave liposomes were simultaneously observed with increased DOX concentrations; however, their morphologies were not readily determined by 2-D cryo-TEM imaging. Precise data analysis of the 3-D parameters of each liposome allowed semi-quantitative evaluation of the transformation of spherical liposomes into non-spherical - prolate, oblate, and concave liposomes.