Endothelial nitric oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. Heat shock protein 90 (HSP90) binds directly to eNOS, augmenting NO production. We have used purified proteins to characterize further the mechanism by which HSP90 increases eNOS activity at low (100 nm) and high (10 microm) Ca(2+) levels. In the presence of calmodulin (CaM), HSP90 increased eNOS activity dose dependently at both low and high Ca(2+) concentrations. This effect was abolished by the specific HSP90 inhibitor geldanamycin (GA) at both calcium concentrations. The EC(50) values of eNOS for both Ca(2+) and CaM were decreased in the presence of HSP90. HSP90 also significantly increased the rate of NADPH-dependent cytochrome c reduction by eNOS at both low and high Ca(2+) concentrations. HSP90 bound to eNOS in a dose-dependent manner, and the amount of bound HSP90 also increased with increasing Ca(2+)/CaM. At 100 nm Ca(2+), HSP90 promoted dose-dependent CaM binding to eNOS that was fully inhibitable by GA. At high calcium, HSP90 did not affect CaM binding to eNOS, but GA inhibited HSP90 binding to eNOS. At high Ca(2+), HSP90 caused the V(max) of eNOS for l-arginine to increase by 2-fold, but the K(m) of eNOS was unchanged. HSP90 bound preferentially to CaM-prebound eNOS and significantly increased both its NO synthesis and reductase activities. These data support that HSP90 promotes eNOS activity by two mechanisms: (i) a CaM-dependent mechanism operative at low Ca(2+) concentrations, characterized by an increase in the affinity of eNOS for CaM and (ii) a CaM-independent mechanism apparent at high Ca(2+) concentrations, characterized by stimulation of eNOS reductase activity without further change in CaM binding. These studies contribute to our understanding of eNOS activation by HSP90 and provide a basis for in vitro studies of other eNOS-interacting proteins.