Summary: The asymmetrical structure of bent immunoglobulin E (IgE) bound to its high-affinity receptor, FcɛRI, suggests a possible role for this configuration in the regulation of signaling mediated by cross-linking of FcɛRI on the surface of mast cells and basophils. Indeed, the presence of bound IgE strongly influences the capacity of cross-linked FcɛRI dimers to trigger mast cell degranulation, implicating orientational constraints by bound IgE. Bivalent ligands that cross-link by binding to bivalent IgE can form linear and cyclic chains of IgE/FcɛRI complexes, and these exhibit only limited capacity to stimulate downstream signaling and degranulation, whereas structurally analogous trivalent ligands, which can form branched networks of cross-linked IgE/FcɛRI complexes, are more effective at cell activation. Long bivalent ligands with flexible spacers can form intramolecular cross-links with IgE, and these stable 1:1 complexes are very potent inhibitors of mast cell degranulation stimulated by multivalent antigen. In contrast, trivalent ligands with rigid double-stranded DNA spacers effectively stimulate degranulation responses in a length-dependent manner, providing direct evidence for receptor transphosphorylation as a key step in the mechanism of signaling by FcɛRI. Thus, studies with chemically defined oligovalent ligands show important features of IgE receptor cross-linking that regulate signaling, leading to mast cell activation.