We have used 5, 12, and 16-nitroxyl stearic acid (5, 12, and 16-NSA) to spin label erythrocytes in order to investigate complement-induced changes in the organization of lipid bilayers. These changes are characterized by measurements of the hyperfine coupling, 2T11, a sensitive indicator of the motional freedom of the spin label and hence a measure of membrane fluidity. When 5, 12, or 16-NSA-labeled E or EA are lysed in hypotonic solution the value of 2T11 decreases indicating an increase in the fluidity of the bilayer. Upon resealing the lysed E or EA in the presence of Mg2+, 2T11 returns to its normal value. The fluidity of EAC ghosts, as measured by 2T11 is comparable to that of hypotonically lysed E or EA. When EAC are resealed in Mg2+ there is a significant decrease in membrane fluidity, for 2T11 increases to values greater than those obtained for resealed ghosts of E and EA.