Previously we have shown that pulmonary arterial endothelial cells (PAEC) isolated from fetal lambs produce significant levels of nitric oxide (NO) on stimulation but minimal superoxide whereas PAEC isolated from 4-week old lambs produce significant amounts of both NO and superoxide. These data indicated that a certain degree of uncoupling of endothelial NO synthase (eNOS) occurs in PAEC during postnatal development. In this study, we sought to extend these studies by investigating the potential role of HSP90 in eNOS coupling. Western blot analyses revealed higher HSP90 expression in PAEC isolated from fetal compared to 4-week old lambs while the analysis of recombinant human eNOS activation in vitro in the presence of HSP90 indicated that HSP90 significantly augmented NO production while inhibiting superoxide generation from eNOS. To further investigate whether HSP90 could be involved in uncoupling of eNOS in PAEC isolated from 4-week old lambs, we utilized an adenovirus to over-express HSP90. We found that over-expression of HSP90 significantly increased the shear stimulated association of HSP90 with eNOS and led to significant increases in NO production and reduced NOS dependent superoxide generation. Conversely, the exposure of PAECs isolated from fetal lambs to the HSP90 inhibitor, radicicol, led to significant decreases in eNOS-HSP90 interactions, decreased shear stimulated NO generation and increased NOS dependent superoxide production indicative of eNOS uncoupling. Finally, we examined eNOS-HSP90 interactions in our lamb model of pulmonary hypertension associated with increased pulmonary blood flow (shunt). Our data indicate that HSP90- eNOS interactions are decreased in Shunt lambs and that this is associated with decreased NO generation and an increase in eNOS-dependent generation of superoxide. Together our data support a significant role for HSP90 in promoting NO generation and inhibiting superoxide generation by eNOS and indicate that the disruption of this interaction may be involved in the endothelial dysfunction associated with pulmonary hypertension.