Polyethyleneglycol (PEG) has often been used for the modification of liposomes, but it is difficult to insert PEG on the surface of liposomes, and the effects of modification are not marked enough. In this study, we examined the fixed aqueous layer thickness (FALT) of single or mixed PEG (molecular weight, 340, 500, 900, 2000)-modified doxorubicin (DOX) liposomes, and physical character and biological properties of these liposomes. On single PEG-modification, as the PEG-molecular weight increased, FALT also increased, but the ratio of the increase was reduced. While on modification by a mixture of PEG2000 and PEG with a short polyoxyethylene chain (PEG340 or PEG500), FALT increased compared with the single PEG2000-modified value. Moreover, when liposomes were modified by mixture of PEG2000 and PEG500, we recognized the most suitable mixed rate (PEG2000, 500=2:1), showed the maximum FALT. On the other hand, in vivo, as increase of FALT, DOX concentrations increased in the plasma and in the tumor, decreased in the liver. Furthermore, liposomes with remarkable increase of FALT showed enhancement of antitumor activity. As a result, the connection among increase of FALT and improvement of circulation in blood, the involvement of antitumor activity of DOX of these liposomes was suggested.