OBJECTIVE Glucocorticoids are the most potent and widely accepted anti-inflammatory agents in the treatment of pathological conditions of the gastrointestinal tract in part by inhibiting the synthesis of proinflammatory prostanoids and leukotrienes. Multiple forms of phospholipase A2 may be associated with the production of these metabolites; this study focused on the molecular mechanism(s) by which glucocorticoids control expression of the arachidonyl-selective, cytosolic phospholipase A2 (cPLA2) in intestinal cells. METHODS Northern analysis, a transcriptional assay, and enzymatic evaluation were used to access expression of the cPLA2 gene in rat small intestinal epithelial and mouse fibroblast cell lines treated with dexamethasone. RESULTS Basal cPLA2 messenger RNA (mRNA) expression was repressed 75% in the presence of dexamethasone with a concomitant decrease in enzymatic activity. Nuclear runoff assays showed a marked decline in de novo cPLA2 RNA synthesis, implicating a transcriptional mechanism associated with the dexamethasone-mediated suppression of cPLA2. Induced expression of cPLA2 mRNA by several proinflammatory cytokines was blocked by cotreatment with dexamethasone. CONCLUSIONS Glucocorticoids are capable of markedly altering basal and cytokine-stimulated cPLA2 gene expression in intestinal epithelial cells, leading to a reduction in arachidonate pools in these cells. Dexamethasone-dependent inhibition occurs through a direct reduction of de novo cPLA2 gene transcription.