Fluorescence lifetime correlation spectroscopy (FLCS) has been used to probe the influence of PEG-8000 on the fluidity of fluorescently labeled planar supported lipid bilayers on ozone plasma treated glass. The lipid membrane compositions examined were; DOPC, DOPC/DOPS (80/20 mol/mol) and DMPC, with and without cholesterol. The lateral diffusion coefficients (D) for supported lipid bilayer films of these layers without cholesterol were 7.9 ± 0.2, 7.9 ± 0.4 and 5.5 ± 0.1 μm2 s-1 respectively. The high fluidity reflected the super-hydrophilicity (contact angle of 0) of the ozone treated plasma glass substrate. Using DOPE conjugated Atto 655 as a probe, exposure of the lipid bilayer to a 30% wt/wt aqueous solution of PEG, followed by washing, dramatically increased the diffusion coefficients of the probe within the film. For example, the diffusion coefficient for the DOPC bilayer increases by nearly an order of magnitude to 51.4 ± 2.6 μm2 s-1. The autocorrelation curves for DOPC/DOPS (80/20 mol/mol) and DMPC bilayers required a two-component model for adequate fit of their behaviour yielding both fast and slow components of the diffusion. In all cases, when hydrophilic DOPE-Atto 655 was used as the probe, treatment of the lipid bilayer with PEG resulted in non-Brownian diffusion. Importantly, the observed diffusion behavior observed depends on the identity of probe. In contrast, when a hydrophobic probe (DOPE-NapthBodipy) was employed PEG showed relatively little impact on the observed diffusion rates. This was attributed to orientation of the reporter probe in the lipid bilayer and its aqueous interface. Specifically, DOPE-Atto 655 is believed to associate strongly with PEG mesh at the aqueous interface of the lipid bilayer, its diffusion strongly influenced by the structure in this region, whereas DOPE-NapthBodipy remains in the interior of the bilayer where it is relatively uninfluenced by PEG. © 2012 The Royal Society of Chemistry.