Cytosolic prostaglandin (PG) E(2) synthase (cPGES) is constitutively expressed in a wide variety of cells and converts cyclooxygenase (COX)-1-derived PGH(2) to PGE(2). Given the fact that cPGES is identical to p23, a heat shock protein 90 (Hsp90)-binding protein, we herein examined the effect of Hsp90 on PGE(2) generation by cPGES. Incubation of cPGES with Hsp90 resulted in a significant increase in PGES activity in vitro. Association of cPGES with Hsp90 was increased in cells stimulated with A23187 or bradykinin, accompanied by concomitant increases in cPGES activity and PGE(2) production. Moreover, treatment of cells with Hsp90 inhibitors, which destabilized the cPGES/Hsp90 complex, reduced cPGES activity and PGE(2) production to basal levels. These results suggest that the regulation of cPGES activity in cells depends on its association with Hsp90 and provide the first line of evidence that eicosanoid biosynthesis is under the control of the molecular chaperone.