Prostaglandin (PG)E₂ acts in an autocrine fashion to suppress proliferation of lung fibroblasts and production of collagen, and may negatively regulate pulmonary fibrosis. The role of Group IVA cytosolic phospholipase A₂ α (cPLA₂α) in PGE₂ production was investigated by comparing lung fibroblasts from wild-type and cPLA₂α-deficient mice. Arachidonic acid release from wild-type mouse lung fibroblasts (MLF^+/+) was stimulated by serum, A23187 plus phorbol-myristate acetate (PMA), and lysophosphatidic acid (LPA) plus platelet-derived growth factor, but was ⩾ 80% lower from cPLA₂α-deficient MLF (MLF^−/−). Transforming growth factor-β increased cyclooxygenase-2 (COX2) expression to similar levels in MLF^+/+ and MLF^−/−, but MLF^+/+ produced an order of magnitude more PGE₂ than MLF^−/− in response to A23187/PMA or platelet-derived growth factor/LPA. MLF^+/+ synthesized less collagen than MLF^−/−, supporting a role for PGE₂ in suppressing collagen production. An SV40 immortalized line developed from MLF^+/+ released arachidonic acid and expressed COX2 to levels similar to those of primary fibroblasts but produced 30-fold lower amounts of PGE₂. Unlike primary fibroblasts, immortalized cells were deficient in microsomal PGE synthase (mPGES) but expressed slightly higher levels of cytosolic PGES. The results demonstrate a primary role for cPLA₂α in providing arachidonic acid for PGE₂ production in mouse lung fibroblasts and support a role for this pathway in regulating collagen production.