AbstractRelease of pro-inflammatory mediators by mast cells is a key feature of allergic disease. The ‘dogma’ is that IgE molecules merely sensitise mast cells by binding FcεRI prior to cross-linking by multivalent allergen, receptor aggregation and mast cell activation. However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known. We show that both monovalent hapten and recombinant SPE-7 IgE Fab inhibit its cytokinergic activity as measured by mast cell degranulation and TNF-α release. Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of ‘free’ SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity. We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains and propose that ‘free’ SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction. Initial formation of a tri-molecular complex (one ‘free’ IgE molecule cross-linking two receptor-bound IgE molecules) leads to capture of further ‘free’ and receptor-bound IgEs to form larger clusters that trigger mast cell activation.