Malondialdehyde (MDA), a by-product of the oxidation of polyunsaturated fatty acids, is strongly cytotoxic. Here we report the in vitro ability of melatonin to protect intact human erythrocytes against the damage induced by the exposure to MDA. MDA at 20 microM caused marked variations in the red blood cell (RBC) membrane. High molecular weight fluorescent adducts were formed within minutes with membrane proteins. A 6-hr incubation led to the oxidation of membrane lipids, as reflected by the formation of conjugated diene (CD) lipid hydroperoxides and oxidation of vitamin E, and to an increase of the high molecular weight fluorescent adducts, which were an indication of MDA finally generated in the cells. Functional damage to the membrane was evident as a leakage of K+ ions into the incubation medium, and an increased resistance to osmotic lysis. A time-dependent hemolysis was observed by exposure of RBCs to 20 microM MDA for 6-12 hr. Melatonin was not a substrate for MDA, therefore it was not able to prevent the early formation of the adducts from the reaction of the MDA in the medium with membrane proteins. Melatonin, however, concentration-dependent prevented the formation of CD lipid hydroperoxides. As a consequence of counteracting the membrane lipid oxidation, the indoleamine prevented the loss of vitamin E and the increase of the fluorescent proteinaceous adducts observed after a 6-hr exposure to MDA. Melatonin also inhibited the K+ loss and returned to normal the osmotic resistance of the erythrocyte in the osmotic fragility test. By protecting membrane lipids and proteins, melatonin effectively prevented the MDA-induced time-dependent hemolysis. In the light of the known radical scavenging properties of melatonin, mechanisms of the cytoprotective effects of melatonin in our system are discussed.