Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E(2) (PGE(2)) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE(2) directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE(2) effect has not been characterized. Our goal is to define the PGE(2) receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE(2) induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP(2) receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE(2)-selective analogs further demonstrates that EP(2) mediates cAMP formation and cystogenesis. Functional evidence for a role of EP(2) receptor in mediating cAMP signaling was also provided by inhibiting EP(2) receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE(2) produced in cyst fluid binds to adjacent EP(2) receptors located on the apical side of cysts and stimulates EP(2) receptor expression. PGE(2) binding to EP(2) receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP(2) receptor in mediating the PGE(2) effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.