Abstract A non-ideal lipid binary mixture (dilauroylphosphatidylcholine/ distearoylphosphatidylcholine), which exhibits gel/fluid phase coexistence for wide temperature and composition ranges, was studied using photophysical techniques, namely fluorescence anisotropy, lifetime and resonance energy transfer (FRET) measurements. The FRET donor, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanolamine, and a short-tailed FRET acceptor, 1,1′-didodecil-3,3,3′,3′-tetramethylindocarbocyanine (DiIC 12(3)), were shown to prefer the fluid phase by both intrinsic anisotropy, lifetime and FRET measurements, in agreement with published reports. The other studied FRET acceptor, long-tailed probe 1,1′-dioctadecil-3,3,3′,3′-tetramethylindocarbocyanine (DiIC 18(3)), is usually reported in the literature as partitioning mainly to the gel. While intrinsic lifetime studies indeed indicated preferential partition of DiIC 18(3) into a rigidified environment, FRET analysis pointed to an increased donor–acceptor proximity as a consequence of phase separation. These apparently conflicting results were rationalized on the basis of segregation of DiIC 18(3) to the gel/fluid interphase. In order to fluid-located donors sense these interphase-located acceptors, fluid domains should be small (not exceed ∼10–15 nm). It is concluded that membrane probes which apparently prefer the gel phase may indeed show a non-random distribution in this medium, and tend to locate in an environment which simultaneously leads to less strict packing constraints and to favorable hydrophobic matching interactions.