The effect of cholesterol on the formation and hydration behavior of solid-supported polysorbate 20 (Tween 20)/cholesterol self-assemblies was investigated by means of in situ energy-dispersive X-ray diffraction in a wide range of relative humidity (0.4 < RH < 1). At low hydration, Tween 20 and cholesterol were found to demix, with the latter molecules forming crystallites with a pseudobilayer structure (d approximately = 34 A). Water adsorption promoted the progressive solubilization of cholesterol crystallites. When in the presence of enough cholesterol, the formation of niosomal bilayer membranes rich in Tween 20 occurred (RH approximately = 0.985). Upon further hydration, two distinct regimes associated with remarkable changes in the niosomal membrane structure were identified. In the first regime (0.985 < RH < 0.988), the swelling of the lamellar d spacing was due to the enlargement of the membrane thickness. In the second regime, the structure of Tween 20/cholesterol membranes was quite insensitive to hydration, and the thickness of the intermembrane water layer increased substantially. Remarkably, the curve of the calculated number of waters per surfactant molecule showed a distinct break at RH approximately 0.988, suggesting that the observed structural change in niosomal membranes was most likely due to the completion of the filling of the Tween 20 hydration shell. At full hydration, niosomal membranes exhibited the same lamellar d spacing of niosomes vesicles in aqueous solution. The process completely reversed upon dehydration.