It is demonstrated that single-molecule tracking of a fluorescently labeled protein undergoing transient binding to model membranes presents a useful method of obtaining fluid properties. The labeled ACBP protein was tracked during its binding to free-standing giant unilamellar vesicles (GUVs) and supported bilayers prepared from the GUVs in the same environment. The analysis of images that are blurred as a result of fast probe diffusion was discussed. An examination of the lateral diffusion trajectories revealed a homogeneous diffusion on the top segments of the GUVs with D = 6.9 +/- 0.3 microm(2)/s. The supported bilayer experiments revealed two diffusion processes, one with Df = 3.1 +/- 0.4 microm(2)/s and the other with Ds = 0.078 +/- 0.001 microm(2)/s. The 2-fold difference in the lipid bilayer mobility for the free-standing and fast components in the supported bilayers is attributed to the known effect of frictional coupling with the solid support. The slow mobile fraction in the bilayer is suggested to be associated with the migration of pore-like structures, originating from the interaction of the membrane with the glass support.