By use of multilamellar phosphatidylcholine (PC) liposomes of different acyl composition and cholesterol content as model membranes, we studied whether or not membrane fluidity affects the assembly process of Staphylococcus aureus alpha-toxin. Under conditions using fluid and solid membranes, we assayed accessibility (or hemolytic activity) of liposome-bound alpha-toxin to rabbit erythrocytes added, hexamerization of membrane-bound toxin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nondenaturating conditions, and susceptibility of liposome-bound toxin to trypsin digestion. Our data indicated 1) that alpha-toxin bound to PC membrane as a hemolytically active monomer (or reversibly bound state); 2) that when the membrane was fluidized either by phase transition of PC or by inclusion of cholesterol over 20 mol %, the hemolytically active monomer of the toxin was irreversibly converted to nonhemolytic monomer (and/or unstable oligomer) in a first-order kinetics with a t1/2 of about 1 min, and thereafter hexamerization of the toxin gradually proceeded in the following 60-90 min; 3) that alpha-toxin might have different topology and/or conformation in PC membrane, depending on the presence or absence of cholesterol in the PC membrane; and 4) that coexistence of unsaturated acyl chain-carrying PC and cholesterol was a prerequisite for efficient hexamerization of alpha-toxin in membrane. Thus, increase in membrane fluidity promoted the assembly process of S. aureus alpha-toxin.