A study was made of the mechanism of hemolysis by cationic surface‐active agents. Phospholipids were found to be released from the cell membrane prior to lysis by the cationic agents under the conditions of physiological pH and ionic strength, indicating an important role of electrostatic interaction between the surface‐active cations and the phospholipid anions in hemolysis. The adsorption experiments showed that the binding of surface‐active cations to the lipoprotein layer of cell membrane is a prerequisite for the hemolysis to take place. The temperature dependence of adsorption suggested a hydrophobic nature of the binding. It was proposed that the adsorbed surface‐active cations make the channels through which the phospholipid molecules are removed out of the cell membrane and that the removal of phospholipids from the lipoprotein layer causes the alteration of protein conformation, thereby allowing the release of hemoglobin into the surrounding medium. Copyright © 1969 Wiley‐Liss, Inc., A Wiley Company