Crosslinking the high affinity IgE receptor, FcϵRI, on basophils and mast cells initiates cascades of biochemical events leading to degranulation, membrane ruffling and other physiological responses. Downstream of FcϵRI and its coupled tyrosine kinases, Lyn and Syk, scores of different proteins and lipids are implicated in these signaling cascades and new players are being identified continuously. Here, we use immunogold probes to label receptors and signaling proteins on the cytoplasmic face of membrane sheets prepared from RBL-2H3 mast cells and transmission electron microscopy to examine their distributions in relationship to each other and to features of the membrane. New topographical data are integrated with existing knowledge of the biochemistry of FcϵRI signaling and of cell shape during signaling to implicate at least two distinct membrane domains in FcϵRI signaling. “Primary signaling domains”, also called osmiophilic patches, are recognized by their dark staining with osmium, adjacency to coated pits (previously mapped to planar membrane between lamellae) and by the characteristic presence of receptor, Syk and PLCγ2, but not Lyn. “Secondary signaling domains” are characterized by the presence of large elliptical linker for activation of T cells (LAT) rafts and of PLCγ1 (previously mapped to lamellae) but not receptor. The signaling proteins, Vav, Grb2, Cbl and Gab2, and the endocytic proteins, AP2 and clathrin, all map to the primary domains, while the p85 regulatory subunit of phosphatidylinositol 3 (PI 3)-kinase maps to both domains. Recognition that FcϵRI signaling is controlled not only by which chemical species are available for interaction, but also by where the interactions occur, may provide new opportunities for the modeling of signaling cascades and new targets for the development of drugs to treat allergies and asthma. [Copyright &y& Elsevier]