In this study, we characterized the encapsulation of amphipathic nystatin into liposomes with or without cholesterol (CH) and a polyethylene glycol derivative, distearoyl-N-(monomethoxy poly(ethylene glycol)succinyl)phosphatidylethanolamine (DSPE-PEG). The highest encapsulation efficacy of nystatin into liposomes (151 microg nystatin/mg lipid) was obtained with a cholesterol-free lipid composition containing 6 mol% of DSPE-PEG. The encapsulation efficacy was decreased by the incorporation of CH and improved by the incorporation of DSPE-PEG. In liposomes composed of dipalmitoylphosphatidylcholine (DPPC)/CH (2:1, mol/mol), the highest encapsulation efficacy of nystatin liposomes (84 microg/mg lipid) was achieved by the addition of DSPE-PEG and hydration with 9% sucrose solution, as compared with 13 microg/mg lipid without DSPE-PEG. The encapsulated amount increased with increasing amount of DSPE-PEG used and plateaued at 6 mol% of DSPE-PEG. The optimum molecular weight of PEG in DSPE-PEG was 2000 and a larger molecular weight resulted in lower encapsulation. The incorporation of CH affected the self-association of nystatin with lipid membranes, which was detected by fluorescence measurement. The molecular interaction between an amino group in nystatin and a phosphate group in DSPE-PEG plays an important role in efficient encapsulation of nystatin. Finally, the encapsulation characteristics of nystatin were compared with those of amphotericin B (AmB). Nystatin more readily associated with CH-free lipid membranes, but, AmB more readily interacted with DSPE-PEG. The results indicated that the differences in the molecular association of AmB or nystatin with lipids or DSPE-PEG are reflected in the encapsulation characteristics in liposomes.