Phospholipase activation has been considered rate-limiting for ligand-stimulated prostaglandin (PG) synthesis. Constitutive prostaglandin synthase EC 1.14.99.1 (PGS-1) has been thought to convert arachidonate released following ligand stimulation to PGH2. However, a second prostaglandin synthase, TIS10/PGS-2, is inducible in many cell types, including fibroblasts and macrophages. We have used antisense oligonucleotide inhibition of TIS10/PGS-2 induction to investigate the role of this enzyme in ligand-induced prostaglandin production in fibroblasts and macrophages. Antisense TIS10/PGS-2 oligonucleotides block mitogen-induced prostaglandin production and TIS10/PGS-2 protein synthesis in both Swiss 3T3 cells and murine embryo fibroblast cultures, without inhibiting either constitutive PGS-1 activity or mitogen-stimulated arachidonic acid release. TIS10/PGS-2 antisense oligonucleotides also block production of PGE2 from endogenous arachidonate following endotoxin stimulation of both RAW 264.7 macrophage cells and murine peritoneal macrophages. In contrast, the constitutive prostaglandin synthase present in macrophages is unable to convert arachidonate released following endotoxin stimulation into prostaglandin. TIS10/PGS-2 expression is necessary for both mitogen-induced prostaglandin production in murine fibroblasts and endotoxin-induced prostaglandin synthesis in macrophages. PGS-1 present in these cells cannot utilize arachidonic acid released in response to mitogen or endotoxin stimulation.