The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside G(M1) into nano- and microscale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of G(M1) allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the colocalization of cholesterol and G(M1) in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d > 3 mu m); (3) nanometer-scale domains (d = 100 nm to 1 mu m) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/G(M1)-rich micrometer-scale phase, while G(M1)-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.