Probe distribution heterogeneity was studied in model membranes (large unilamellar vesicles of dipalmitoylphosphatidylcholine) using resonance energy transfer. Two Forster pairs were used: octadecylrhodamine B (ORB; donor)/1,1',3,3,3',3'-hexamethylindotricarbocyanine (DiIC(1)(7); acceptor) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine (NBD-PE; donor)/N-(lissamine-rhodamine B)-dipalmitoylphosphatidylethanolamine (Rh-PE; acceptor). The donors' fluorescence decays were analyzed in the framework of the mean local concentration model (see preceding article). As acceptor concentration distributions, discrete, Gaussian, and sum of two Gaussian functions were considered. In the fluid phase (50 degrees C) for moderate (<1%) acceptor concentrations, both donors and accepters of the two pairs are essentially randomly distributed, and the recovered acceptor concentration distribution curves are well described by single narrow Gaussians. For the gel phase (25 degrees C), a sum of two Gaussians acceptor concentration distribution is necessary to describe the data. It is concluded that, in the gel phase, for the ORB/DiIC(1)(7) pair, accepters partially segregate into a pseudo (defect) phase and donors are randomly dispersed in the bulk lattice. At variance, for the NBD-PE/Rh-PE pair, partial segregation of both probes into a pseudo (defect) phase occurs at 25 degrees C. These conclusions are supported by detailed additional photophysical measurements for these probes in membranes (steady-state energy transfer, fluorescence self-quenching in steady and transient states, and energy migration), and agree with the preceding article's simulations.