Both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been implicated in the biliary tract carcinogenesis. However, it is not known if these inflammatory mediators are induced by interdependent or parallel pathways. Because iNOS activity has been associated with diverse gene expression, the aim of this study was to determine if iNOS induces COX-2. To address this objective, immortalized, but nonmalignant, murine cholangiocytes, 603B cells were employed for these studies. Both iNOS and COX-2 protein and mRNA were both expressed in these cells. However, iNOS inhibition with either 1400W or stable transfection with an iNOS antisense construct inhibited COX-2 mRNA and protein expression, an effect which was reversed by NO donors. COX-2 mRNA expression in 603B cells was reduced by pharmacologic inhibitors of the p38 mitogen-activated protein kinase (MAPK) and Janus-N-terminal kinase (JNK1/2) pathways. In contrast, neither inhibitors of the soluble guanylyl cyclase inhibitor/protein kinase G nor p42/44 MAPK pathways attenuated COX-2 mRNA expression. Finally, 603B cells grew at a rate 3-fold greater than 603B-iNOS antisense cells. The low growth rate of 603B-iNOS antisense cells could be restored to near that of the parent cell line with exogenous prostaglandin E₂. In conclusion, iNOS induces COX- 2 expression in cholangiocytes, which promotes cell growth. COX-2 induction may contribute to iNOS associated carcinogenesis.