Sendai F-virosomes, a novel type of liposome with reconstituted Sendai F-proteins, have been tested as a delivery system for various bioactive materials. However, encapsulation limitations and difficulties in controlling their constituents were drawbacks for further application to therapeutic purposes. We have tried to control virosomal constituents and have enhanced drug encapsulation efficiency into the virosomes. In vitro cytotoxicity of doxorubicin encapsulated in the F-virosomes were compared with free doxorubicin and doxorubicin in conventional liposomes. The F-virosomes were spontaneously prepared by detergent dialysis, a reconstitution process of Sendai F-proteins into liposomes. The reconstitution density of F-proteins affected the vesicle size of virosomes prepared by detergent dialysis; the larger amount of F-proteins made a smaller size of virosomes. There was little variation of size with time at physiological conditions, whilst the vesicle size of virosomes increased at acidic storage conditions (pH 5.5). Doxorubicin encapsulated in the F-virosomes exhibited a lower IC50 against B16BL6 mouse melanoma cells and Chang human hepatocarcinoma cells than that in conventional liposomes. The F-virosomes also exhibited higher cellular uptake than conventional liposomes. Addition of dioleoylphophatidylethanolamine, a fusogenic phospholipid, into the F-virosome further increased the cellular uptake as well as in vitro cytotoxicity. These types of virosome formulations can be clinically applicable as versatile vesicles for the efficient delivery of various therapeutic drugs, including genetic materials.