Liposomes have been widely used delivery systems, particularly relevant to the development of cancer therapeutics. Numerous liposome-based drugs are in the clinic or in clinical trials today against multiple tumor types; however, systematic studies of liposome interactions with solid or metastatic tumor nodules are scarce. This study is describing the in vitro interaction between liposomes and avascular human prostate (LNCaP-LN3) tumor spheroids. The ability of fluorescently labelled liposomal delivery systems of varying physicochemical characteristics to penetrate within multicellular tumor spheroids has been investigated by confocal laser scanning microscopy. A variety of liposome characteristics and experimental parameters were investigated, including lipid bilayer composition, duration of liposome-spheroid interaction, mean liposome size, steric stabilization of liposomes.