The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.