The regulation of expression of cyclooxygenase 2 (COX-2) was investigated by treatment with PGE₂ in human endometrial adenocarcinoma cell line HEC-1B. One μM PGE₂ could stimulate the expression of COX-2 approximately twofold in this cell line. The same concentration of PGE₂ also stimulated activation of mitogen-activated protein kinase (MAP kinase) and protein kinase B (PKB). PGE₂-induced MAP kinase activation was sensitive to a MAP kinase kinase (MEK) inhibitor, PD098059, and a protein kinase A inhibitor, H-89. PD098059 and H-89 also partially inhibited the expression of COX-2 stimulated by PGE₂. PGE₂ could stimulate the activation of PKB, which was sensitive to phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. Whereas wortmannin alone partially inhibited the expression of COX-2, a combination of wortmannin and PD098059 totally inhibited PGE₂-mediated COX-2 expression. These results suggest that MAP kinase and PI3K pathways are stimulated with PGE₂, and that both of these pathways are involved in the expression of COX-2. In addition, they also suggest that protein kinase A remains upstream of PGE₂-induced activation of MAP kinase in HEC-1B cells.