Using atomic force microscopy and molecular force probe, we have studied the structure and nanomechanical response to nano-indentation of multilayered films of dioleoylphosphatidylcholine films prepared by solution spreading, spin-coating, and capillary methods. The influence of the film thickness on the apparent elastic (Young's) modulus is investigated. Young's moduli of samples prepared by the different methods were found to be similar for sufficiently thick films. An ostensible influence of substrate coupling on the apparent Young's modulus is observed for thinner lipid films. The results are discussed in comparison with previously reported estimates for supported lipid bilayers (membranes) and cells. (C) 2008 Elsevier B.V. All rights reserved.