The effects of 2,6-di-O-methyl-alpha-cyclodextrin (DM-alpha-CyD) on hemolysis and morphological changes in rabbit's red blood cells (RBC) were examined, compared with those of alpha-cyclodextrin (CyD) and 2-hydoxypropyl-alpha-cyclodextrin (HP-alpha-CyD). The hemolytic activity of alpha-CyDs increased in the order of HP-alpha-CyD<alpha-CyD<DM-alpha-CyD. The three alpha-CyDs induced morphological changes of RBC from discocyte to stomatocyte. At the same concentration (3mM) of alpha-CyDs, DM-alpha-CyD and alpha-CyD released phospholipids, rather than cholesterol, and DM-alpha-CyD markedly released proteins from RBC membranes, compared to alpha-CyD and HP-alpha-CyD. The treatment of RBC with DM-alpha-CyD lowered the extent of a fluorescent sphingomyelin analogue from lipid rafts of RBC membranes in a concentration-dependent manner. These results suggest that DM-alpha-CyD has higher hemolytic and morphological change activity than alpha-CyD and HP-alpha-CyD through more extraction of phospholipids including sphingomyelin and proteins, not cholesterol, from RBC membranes than alpha-CyD and HP-alpha-CyD.