In mast cells, basophils, and the RBL-2H3 tumor mast cell model, crosslinking cell surface IgE-receptor complexes by multivalent ligands activates a signal transduction pathway that leads to the secretion of histamine, serotonin, and other inflammatory mediators. Receptor crosslinking in RBL-2H3 cells also changes cell surface morphology and increases F-actin assembly. Previously, Robertson et al.(J. Immunol., 135: 4565–4572, 1986) demonstrated that crosslinked IgE-receptor complexes become associated with the Triton X-100-insoluble fraction (the “cytoskeleton”) of RBL-2H3 cells and raised the possibility that receptor-cytoskeletal association may be a required step in the stimulation of secretion. The studies reported here confirm by flow cytometry that crosslinking cell surface IgE by antigen induces the association of the crosslinked complexes with the detergent-insoluble fraction. Dose-response studies, also reported here, indicate that the detergent insolubility of the complexes does not correlate with secretion. Thus, secretion increases with antigen concentration to a maximum beyond which more antigen causes less, not more, secretion. There is little residual detergent-insoluble IgE at the concentrations of antigen that promote optimal secretion, whereas the association of IgE with the detergent-insoluble fraction is maximal at the high antigen concentrations that result in reduced secretion. The addition of monovalent hapten to reduce the amount of crosslinking caused by high concentrations of antigen increases secretion and simultaneously reduces the association of IgE with the detergent-insoluble fraction. Dihydrocytochalasin B, an inhibitor of antigen-stimulated actin polymerization, also increases the rate and extent of secretion and simultaneously delays the association of crosslinked IgE-receptor complexes with the detergent-insoluble fraction. From these data, we propose that the association of crosslinked IgE receptors with the detergent-insoluble fraction of RBL-2H3 cells increases with increased receptor crosslinking, is enhanced by antigen-induced actin polymerization, and is more likely related to the termination than the stimulation of secretion. The ligand-induced conversion of receptors to a detergent-insoluble form is not restricted to mast cells but occurs in a variety of cell types. Its general function may be to limit the generation or transmission of transmembrane signals.