We have determined the membrane topography of the high-affinity IgE receptor, FcεRI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017–1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcεRI and Lyn are distributed as small clusters (2–9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcεRI clusters contain Lyn. In contrast, there is essentially no FcεRI-Syk colocalization in resting cells. 2 min after FcεRI cross-linking, ∼10% of Lyn colocalizes with small and medium-sized FcεRI clusters (up to 20 gold particles), whereas ∼16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20–100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcεRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcεRI signaling.