Red blood cell deformability is important for effective circulation in the capillaries. It is known that red cell deformability is significantly reduced during septic shock. Surface to volume ratio, physical effects of the cytoskeletal proteins and the fluidity of lipid bilayer are some of the important intrinsic factors that regulate this mechanical function. Alterations in the physical conformation of cytoskeletal proteins in septic conditions could significantly alter their function. In this study, erythrocytes in whole blood were treated with lipopolysaccharide, the outer covering of Gram-negative bacteria released during Gram-negative sepsis. Electron paramagnetic resonance spectroscopy in conjunction with a protein-specific maleimide nitroxide spin label covalently bound to cytoskeletal proteins was used to investigate the resulting changes occurring in the physical state of cytoskeletal proteins in isolated membranes. Treatment of red blood cells with a lipopolysaccharide concentration as low as 40 micrograms/mL of blood solution for 90 minutes showed a significant decrease in the relevant EPR parameter (p < 0.01) of the spin label bound to subsequently isolated membranes, suggestive of a decreased segmental motion of the spin label and an increase in cytoskeletal protein-protein interactions. These results suggest a marked conformational alteration in the cytoskeletal proteins induced by the lipopolysaccharide and may explain, in part, the marked reduction in red blood cell deformability during septic shock. Bacterial lipopolysaccharide does not exert most of its effects on the host directly, but rather elicits the production of host factors that leads to complex septic shock. Leukocytes, endothelial tissue and many other cells release these mediators. Leukocytes are thought to be a particularly important source of such mediators, including cytokines (tumor necrosis factor, interleukins, etc.), oxygen free radicals, proteases, and hydrolyses. In order to characterize the possible mechanism by which the lipopolysaccharide acts on the physical state of the erythrocyte cytoskeleton, erythrocytes void of leukocytes and plasma were treated with lipopolysaccharide. The relevant EPR parameter showed no significant change over the control value. These results indicate that the leukocytes and their factors are responsible for the rearrangements seen in the cytoskeletal proteins of the erythrocyte membrane.