The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-b-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin phase maps corresponding to the traditional solid-ordered and liquiddisordered phases and two types of liquid-ordered behavior. Physical properties were less diverse in the mixed phospholipid vesicles, as expected, based on previous studies. Erythrocytes displayed five regions of different combinations of membrane properties along the phase map. Some of the observations identified similarities between the cells and liquidordered behavior observed in the various types of liposomes as well as some interesting