A new hydration cell has been constructed that allows to fully hydrate aligned lipid multibilayers as well as to study lipid hydration by using in situ time-resolved energy dispersive X-ray diffraction. The cell has been used to investigate the evolution of the lamellar d-spacing of dioleoylphosphocoline oriented membranes as a function of both relative humidity (RH) and time. The extreme sensitivity of d to RH was shown and two hydration regimes were detected. (c) 2005 Elsevier B.V. All rights reserved.