The balance of nitric oxide (.NO) and superoxide anion (O(2)) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates.NO. As eNOS is capable of generating both.NO and O(2), we hypothesized that Hsp90 might also mediate eNOS-dependent O(2) production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 microg/ml; 17.8 microm) and then stimulated with the calcium ionophore, (5 microm). GA significantly decreased -stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased -stimulated eNOS-dependent O(2) production (p < 0.001, n = 8). increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent.NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O(2) production.