The distribution of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene between coexisting gel and fluid phospholipid phases in multilamellar vesicles has been examined using fluorescence quenching by spin-labeled phosphatidylcholine. For both thermally-induced and Ca2+-induced lipid phase separation, the ratio of probe concentration in the fluid liquid-crystal phase to that in the gel phase is found to be independent of either the probe concentration or the relative amounts of gel and fluid lipid phases, and hence is an equilibrium concentration ratio, or partition coefficient.