cDNAs were recently isolated from BALB/c mouse mast cells (MCs) that encode the new signaling protein mouse Ras guanine nucleotide releasing protein 4 (mRasGRP4). The present study evaluates the expression pattern and biological activity of mRasGRP4 in a variety of mouse strains. As assessed immunohistochemically and by RNA analysis, mRasGRP4 is not coordinately expressed with any of its family members. Normally, mRasGRP4 is an MC-restricted protein in tissues, and kinetic studies revealed that mRasGRP4 is expressed relatively early in developing MCs. The expression of mRasGRP4 in the fetus before granulated MCs become abundant supports the conclusion that RasGRP4 participates in MC-specific differentiation pathways. Functional studies conducted with recombinant material revealed that mRasGRP4 is a cation-dependent, diacylglycerol (DAG)-regulated, guanine nucleotide exchange factor. Immunoelectron microscopic studies revealed that mRasGRP4 resides in either the cytosol or inner leaflet of the plasma membrane of the MC, implying that DAG controls the intracellular movement of this signaling protein in c-kit-stimulated MCs. The mRasGRP4 gene resides on chromosome 7B1 within a site that is prominently linked to baseline airway reactivity in backcrossed C3H/HeJ and A/J mice. A truncated isoform of mRasGRP4 that lacks its DAG-regulatory domain was isolated from C3H/HeJ mouse MCs. Sequence analysis showed that this isoform is the result of defective splicing of the precursor transcript. MCs play a central role in allergic inflammation. The discovery of a novel isoform of mRasGRP4 in hyporesponsive mice suggests that airway reactivity is influenced by RasGRP4-dependent signaling events in pulmonary MCs.