The effects of ethanol on hemolysis of human red blood cells (RBCs) were studied at 21 +/- 1 degrees C in the saline buffer (138 mM NaCl, 6.1 mM Na2HPO4, 1.4 mM NaH2PO4, 5 mM glucose and pH 7.4). The hemolysis process for ethanol-treated RBCs was preceded by the leakage of the small cation K+ from the cells indicating the colloid-osmotic nature of lysis. Since the extent of membrane lesion increased with an increasing ethanol concentration, osmotic protection experiments by using solutes varying in size were carried out to estimate the diameter of the pore. Quantitative analysis of the data by considering the effect of molecular seiving of the protectants with different sizes indicated that ethanol induced formation of membrane pores with a diameter of approximately 13 A. There was no detectable release of membrane fragments as assayed by the acetylcholinesterase activity, but the membrane structures were significantly perturbed, presumably at the membrane cytoskeletal protein, as evidenced by the altered rheological properties of RBC in the presence of ethanol. It is suggested that the creation of membrane pores might involve in the deranged cytoskeletal network of ethanol-treated RBC.