Previously, we demonstrated that hepatocyte growth factor (HGF) potently stimulates endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production through a calcium- and Akt-mediated phosphorylation at Ser-1179 (Ser-1177 human) in bovine aortic endothelial cells. The regulation of eNOS, however, also involves interaction with chaperone proteins such as heat shock protein (HSP) 90, which can be enhanced by agonist stimulation of the enzyme. In the present work, the role of HSP90 in HGF stimulation of eNOS was examined in an endothelial cell culture system. Treatment of endothelial cells with geldanamycin, a commonly used HSP90 inhibitor, augmented HGF-stimulated eNOS phosphorylation at Ser-1179, while it did not alter eNOS phosphorylation at Thr-497. However, other HSP90 inhibitors, namely 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and radicicol, did not possess similar effects. Neither HGF nor geldanamycin treatment, independently or in combination, altered HSP90/eNOS interaction in endothelial cells. In addition, geldanamycin treatment did not enhance the HGF-induced phosphorylation of Akt, ERK1/2 and p38MAPK. Src kinase inhibition by PP2 also failed to block the geldanamycin effects. These results suggest that geldanamycin, but neither 17-AAG nor radicicol, may enhance HGF-mediated eNOS Ser-1179 phosphorylation by some as yet unknown mechanisms independently of HSP90 inhibition.