Recent studies show that markers for lipid rafts are among the plasma membrane components most likely to be internalized independently of clathrin-coated pits, and there is evidence to suggest that lipid rafts may play a functional role in endocytic trafficking [1-5]. However, lipid rafts themselves are commonly defined purely in biochemical terms, by resistance to detergent extraction. The existence of rafts in live-cell membranes remains controversial [6-8], and their distribution relative to endocytic machinery has not been investigated. This study employs fluorescence resonance energy transfer (FRET) to show that in the plasma membrane (PM) of living cells the glycosphingolipid GM1, labeled with cholera toxin B subunit (CTB) [9,10], is found at least in part within clusters that also include GPI-linked proteins. These clusters are cholesterol-dependent and exclude non-raft proteins such as transferrin receptor and so possess predicted properties of lipid rafts. This type of lipid raft is largely excluded from clathrin-positive regions of the PM. They are found within Caveolin-positive regions at the same concentration as at the rest of the cell surface. The data provide evidence for a model in which lipid rafts are distributed uniformly across most of the PM of nonpolarized cells but are prevented from entering clathrin-coated pits.