A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hall-mark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with inhibitor kappa B kinases (, and ) in non-vascular cells. In this study, we have investigated the interaction of Hsp-90-IKK in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKK interacts with Hsp-90 and this interaction is augmented by HG in endothelial cells. HG also augments transcriptional (3.5±0.65| —folds) and translational (1.97 ± 0.17 —fold) expression as well as the catalytic activity of IKK (2.45± 0.4—folds). Another novel finding is that both IKK and eNOS could be co-immunoprecipitated with Hsp-90 thus indicating the possible existence of a complex of IKK and eNOS interacting with single pool of Hsp-90. Inhibition of Hsp-90 with geldanamycin (2uM) or Radicicol (20 uM) mitigated HG induced-IKK activity. Blocking of IKK expression by IKK inhibitor II (15uM wedelolactone) or siRNA improved NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.