Z-scan fluorescence correlation spectroscopy (FCS) uses multiple focus planes to determine diffusion coefficients in supported phospholipid bilayers (SPBs) without the need for extrinsic calibration. The approach can be used to discriminate diffusion modes. In this work, Z-scan FCS was applied to study the diffusion of the fluorescent lipid analogue DiI-C-18(5) in the top membrane of giant unilamellar vesicles (GUVs) in the electroformation chamber. In contrast to SPBs, the intrinsic estimate of the radial waist might differ from the one obtained by extrinsic calibration. We describe a pragmatic approach to verify the validity of the Z-scan approach under these conditions. (C) 2008 Elsevier B.V. All rights reserved.