Clustering the mast cell function-associated antigen (MAFA) has earlier been shown to inhibit mast cells' secretory response to the type 1 Fcepsilon receptor (FcepsilonRI) stimulus. MAFA is a type II membrane glycoprotein first identified on rat mast cells and contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytosolic domain. This inhibition is induced already upon clustering MAFA alone. Still, the inhibitory capacity of MAFA-FcepsilonRI co-clustering has recently been characterized and co-clustered MAFA molecules were found to exhibit a markedly higher inhibition capacity than MAFA-clusters alone. We have now compared the inhibitory capacity of FcepsilonRI co-clustered MAFA on the secretory response of rat mucosal-type mast cells (RBL-2H3 line) expressing different levels of this inhibitory protein. Reacting these cells carrying an IgE class, 2,4 dinitrophenyl (DNP)-specific monoclonal antibody with DNP-conjugated F(ab')2 fragments of non-specific polyclonal mouse IgG causes clustering of the FcepsilonRI-IgE. Reaction of these cells with DNP-conjugated F(ab')2 fragments of the MAFA-specific, monoclonal antibody G63 co-aggregates MAFA together with the FcepsilonRI-IgE thereby producing FcepsilonRI-IgE-MAFA co-clusters. Results of measurements of the secretory responses of RBL-2H3 cells expressing higher or lower MAFA levels than those of unmodified cells provided further support to the notion that co-clustered MAFA molecules exhibit a markedly higher inhibition capacity than MAFA-clusters alone. The molecular basis for this enhanced inhibition is most probably the increased concentration of the inhibitory cell components in the immediate proximity of the co-clustered FcepsilonRI-MAFA.