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The interaction of beta-cyclodextrin (beta-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after beta-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by beta-CD, indicating that beta-CD is unable to remove sphingomyelin or complexed Chol. However, beta-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by beta-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of beta-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by beta-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)