The mechanism of thermohaemolysis above 48 o C was recently revealed as being of colloidosmotic type. The permeability barrier damage responsible is insensitive to the displacement of cytosol by isotonic NaCl/sucrose media and is related to a thermally induced event in the erythrocyte membrane (EM) at 62 o C, which presumably involves heat alteration of membrane proteins, excluding spectrin and the anion channel. The onset temperature of this event is linearly reduced by ethanol in intact cells and resealed ghosts with sensitivity exceeding 3.5 times that of heat denaturation of spectrin. Thus, at a specific concentration of ethanol (18% v/v), the permeability breakdown event occurs at 39 o C, which precedes the spectrin denaturation by 6 o C, a result which was used here independently to induce and study this event. The event was induced by heating intact cells or resealed ghosts for 3 min in the presence of 18% ethanol at 39.5 o C. Using thermal gel analysis it was established that this event was not accompanied by an intermolecular disulfide bond aggregation of membrane proteins, a phenomenon which usually follows the heat denaturation of proteins. The membranes of cells and ghosts, exposed to different temperatures for 3 min in the presence of 18% (v/v) ethanol, were subjected to SDS-PAGE after modification with the bifunctional amino reagent FNPS (4,4'-difluoro-3,3'-dinitrodiphenylsulfone). The electrophoretic profile obtained strongly depended on the temperature of exposure. If the latter was between 20 and 35 o C, the gel pattern did not differ from that of the intact EM, but if this temperature coincided with the onset temperature (39 o C), the gel profile indicated the occurrence of strong cross-linkage of membrane proteins. These results showed that the 62 o C membrane event involved only a limited conformational change in EM proteins, possibly in some intrinsic ones, during which many reactive groups appeared at a distance, allowing cross-linking by a bifunctional agent but not a direct aggregation of the altered proteins.