Direct visualization of raft-like lo (liquid-ordered) domains in model systems and cells using microscopic techniques requires fluorescence probes with known partitioning preference for one of the phases present. However, fluorescent probes may display dissimilar partitioning preferences in different lipid sys-tems and can also affect the phase behaviour of the host lipid bilayer. Therefore a detailed understanding of the behaviour of fluorescent probes in defined lipid bilayer systems with known phase behaviour is essential before they can be used for identifying domain phase states. Using giant unilamellar vesicles composed of the ternary lipid mixture DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)/cholesterol, for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned either on the basis of quantification of the domain area fractions or by using a well-characterized ld (liquid-disordered)-phase marker. Fluorescent probes were examined both individually and using dual or triple labelling approaches. Most of the probes partitioned individually into the ld phase, whereas only NAP (naphtho[2,3-a]pyrene) and NBD-DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl] preferred the lo phase. We found that Rh-DPPE (Lissamine™ rhodamine B–1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, Tmix. Interestingly, the partitioning of DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy®-PC [2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine]. The specific use of each of the fluorescent probes is determined by its photostability, partitioning preference, ability to detect lipid phase separations and induced change in Tmix. We demonstrate the importance of testing a specific fluorescent probe in a given model membrane system, rather than assuming that it labels a particular lipid phase.