Subepithelial myofibroblast-derived prostaglandin E₂ (PGE₂) regulates epithelial chloride secretion in the intestine. Thrombin is elevated in inflammatory conditions of the bowel. Therefore, we sought to determine a role for thrombin in regulating PGE₂ synthesis by colonic myofibroblasts. Incubation of cultured CCD-18Co colonic myofibroblasts with thrombin, the proteinase-activated receptor 1 (PAR₁)-activating peptide (Cit-NH₂), and peptides corresponding to 2 noncatalytic regions of thrombin (TP367 and TP508) for 18 h increased both cyclooxygenase (COX)-2 expression (immunocytochemistry) and PGE₂ synthesis (enzyme immunoassay). Inhibition of thrombin by D-Phe-Pro-Arg-chloromethylketone (PPACK) did not significantly reduce PGE₂ synthesis, which remained elevated compared with control. We also investigated the basic fibroblast growth factor (bFGF) dependence of thrombin-induced PGE₂ elevations. Recombinant human bFGF concentration dependently increased PGE₂synthesis, and a bFGF neutralizing antibody inhibited PGE₂synthesis induced by TP367 and TP508 (∼40%) and by thrombin (∼20%) (but not Cit-NH₂). Thrombin, therefore, upregulates COX-2-derived PGE₂ synthesis by both catalytic cleavage of PAR₁ and bFGF-dependent noncatalytic activity. This presents a novel mechanism by which intestinal myofibroblasts might regulate epithelial chloride secretion.