Cytosolic phospholipase A (cPLA(2alpha)) catalyzes the formation of arachidonic acid in prostaglandin synthesis. In contrast to the well-described down-regulation of cPLA(2alpha), up-regulation of cPLA(2alpha) by glucocorticoids has been reported in human amnion fibroblasts, which may play a key role in parturition. The mechanisms underlying this paradoxical induction of cPLA(2alpha) by glucocorticoids remain largely unknown. Using cultured human amnion fibroblasts, we found that the induction of cPLA(2alpha) by cortisol required ongoing transcription and synthesis of at least one other protein. The induction of cPLA(2alpha) by cortisol was abolished by mutagenesis of a glucocorticoid response element (GRE) in the promoter. The same GRE was found mediating the classical inhibition of cPLA(2alpha) expression by cortisol in human fetal lung fibroblasts (HFL-1). Cortisol increased Galpha(s) expression in amnion fibroblasts but not in HFL-1 cells. Inhibition of Galpha(s) with NF449 attenuated the phosphorylation of cAMP response element-binding protein-1 (CREB-1) and the induction of cPLA(2alpha) by cortisol in amnion fibroblasts. Both glucocorticoid receptor (GR) and CREB-1 were found bound to the GRE upon cortisol stimulation of amnion fibroblasts. The induction of cPLA(2alpha) by cortisol was blocked by GR antagonist RU486 or protein kinase A inhibitor H89 or dominant-negative CREB-1. In conclusion, cortisol activates the cAMP/protein kinase A/CREB-1 pathway via Galpha(s) induction, and the phosphorylated CREB-1 interacts with GR at the GRE to promote cPLA(2alpha) expression in amnion fibroblasts.