IgE-binding protein (epsilon BP) is a beta-galactoside-binding animal lectin identified by its affinity for IgE. We have reported that epsilon BP also binds the mast cell high-affinity IgE receptor (Fc epsilon RI), via lectin-carbohydrate interaction. We have now studied the physiological significance of epsilon BP-IgE-Fc epsilon RI interactions in mast cell activation using rat basophilic leukemia (RBL) cells as the model system. We report here that both unsensitized and IgE-sensitized RBL cells are activated upon exposure to epsilon BP-coated surfaces. Activation of RBL cells by the lectin epsilon BP can be significantly inhibited by appropriate saccharides. Exposure of RBL cells to epsilon BP-coated surfaces caused cell spreading similar to that caused from adherence to fibronectin-coated surfaces. However, epsilon BP by itself caused mediator release whereas fibronectin only potentiated antigen-mediated activation of RBL cells. Under appropriate conditions, epsilon BP, therefore, has the potential to activate mast cells culminating in augmentation of an inflammatory response.