Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is one of the major events leading to parturition. In marked contrast to its well-described antiinflammatory effect, glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression in human amnion fibroblasts. The mechanisms underlying this paradoxical induction of COX-2 by glucocorticoids have not been resolved. Using cultured human amnion fibroblasts, we found that the induction of COX-2 mRNA expression by cortisol was a glucocorticoid receptor (GR)-dependent process requiring ongoing transcription. Upon transfection of a COX-2 promoter-driven reporter gene into the amnion fibroblasts, cortisol stimulated the COX-2 promoter activity. This was abolished by mutagenesis of a cAMP response element (CRE) at -53 to approximately -59bp as well as by cotransfection of a plasmid expressing dominant-negative CRE-binding protein (CREB). The phosphorylation level of CREB-1 was significantly increased by cortisol treatment of the amnion fibroblasts, whereas the effect was attenuated either by the protein kinase A inhibitor H89 or the p38 -MAPK inhibitor SB203580. The induction of the COX-2 promoter activity and the phosphorylation of CREB-1 were also blocked by the GR antagonist RU486. Chromatin immunoprecipitation (ChIP) assay revealed that the binding of CREB-1 to the CRE of the COX-2 promoter was increased by cortisol treatment of the amnion fibroblasts. In conclusion, cortisol, via binding to GR, stimulated COX-2 expression by increasing phosphorylated CREB-1 binding to the CRE of the COX-2 gene. Cortisol may phosphorylate CREB-1 by activating either protein kinase A or p38-MAPK in the amnion fibroblasts.