The distribution of glycolipid GM1 in supported phospholipid monolayers of a ternary lipid mixture has been studied by near-field scanning optical microscopy (NSOM). Monolayers of equimolar amounts of sphingomyelin, dioleoylphosphatidylcholine, and cholesterol show clear phase separation to give large condensed domains surrounded by a fluid phase, as visualized by both atomic force microscopy (AFM) and addition of a Texas Red labeled lipid that localizes preferentially in the fluid phase. A combination of AFM and one- and two-color NSOM experiments indicates that addition of GM1-Bodipy results in small glycolipid domains within the fluid phase. This is in contrast to previous results demonstrating that GM1 is localized in the condensed phase for similar lipid mixtures and is attributed to dye-induced changes in the distribution of the glycolipid. However, NSOM experiments for GM1-containing monolayers doped with low loadings of GM1-Bodipy resulted in the observation of small fluorescent microdomains within the condensed phase. The high spatial resolution of NSOM allows the detection of small domains that have not been observed previously by fluorescence and demonstrates that similar, although not identical, distributions of glycolipid are detected by AFM and fluorescence. The relevance of these results to rafts in natural membranes is discussed.