Mast cells (MCs) are a major source of prostaglandin (PG) D(2) in connective tissues, and the expression of this eicosanoid has been linked to asthma and other inflammatory disorders. While it is known that the surface receptor c-kit controls PGD(2) expression in MCs by regulating the levels of a synthase that converts PGH(2) to PGD(2), the intracellular signaling proteins that act downstream of c-kit in this cyclooxygenase pathway have not been identified. We recently cloned a new cation-dependent, guanine nucleotide exchange factor/phorbol ester receptor (designated RasGRP4) that is required for the efficient expression of granule proteases in the human MC line HMC-1. GeneChip analysis of approximately 12,600 transcripts in RasGRP4(-) and RasGRP4(+) HMC-1 cells revealed a >100-fold difference in the levels of hematopoietic PGD(2) synthase mRNA. No other transcript in the eicosanoid pathway was influenced by RasGRP4 in a comparable manner. As assessed by SDS-PAGE immunoblot analysis, RasGRP4(+) HMC-1 cells contained substantial amounts of PGD(2) synthase protein. RasGRP4(+) MCs also produced approximately 15-fold more PGD(2) than did RasGRP4(-) MCs when both cell populations were activated by calcium ionophore. The induced transcript is therefore translated, and substantial amounts of functional PGD(2) synthase accumulate in RasGRP4(+) MCs. In support of the conclusion that RasGRP4 controls PGD(2) expression in MCs, inhibition of RasGRP4 expression in the rat MC line RBL-2H3 using a siRNA approach resulted in low levels of PGD(2) synthase protein.