Activation of MMC-34 cells, a murine mast cell line, or bone marrow-derived mast cells by aggregation of IgE cell surface receptors or addition of calcium ionophore stimulates prostaglandin (PG) D2 synthesis and secretion. An initial rapid burst of PGD2 synthesis, complete within 30 min, is followed by a slower subsequent production of PGD2 that reaches a maximum 4 to 8 h after activation in MMC-34 cells. PG synthase 1 (PGS-1) message and protein are expressed constitutively in MMC-34 cells and are not modulated by exposure to calcium ionophore or aggregation of IgE receptors. In contrast, activation of MMC-34 or bone marrow-derived mast cells induces expression of the PG synthase 2 (PGS-2) gene. PGS-2 induction following mast cell activation is blocked by dexamethasone. The initial PGD2 burst in activated MMC-34 cells is prevented by aspirin pretreatment, suggesting that constitutive PGS-1 present in mast cells before activation is responsible for the early PGD2 production in response to activation. In contrast, the later phase of PGD2 production is blocked by dexamethasone, cycloheximide, or NS-398, a PGS-2-specific nonsteroidal anti-inflammatory drug that inhibits PGS-2 enzyme activity but not PGS-1 activity. These data demonstrate that mast cell activation results 1) in the induction of PGS-2 gene expression, and 2) in both PGS-1-dependent PGD2 synthesis and PGD2 synthesis that is dependent on the activation-induced synthesis and activity of PGS-2.