Heat-shock protein 90 (hsp90) has been shown to facilitate neuronal NO synthase (nNOS, type 1) activity in vivo. But the direct effect of hsp90 on purified nNOS has not been determined yet. Moreover, the mechanism underlying the action of hsp90 is not known. nNOS activity is primarily initiated and regulated by the binding of Ca2+/calmodulin (CaM). Therefore, we explored whether hsp90 modulates nNOS activity by affecting CaM binding. Recombinant rat nNOS was purified from the stably transfected cells by affinity chromatography. hsp90 increased nNOS activity in a dose-dependent manner with an EC50 of 24.1±6.4nM. In the presence of hsp90, the CaM-nNOS dose-response curve was shifted markedly to the left and the maximal activity was also elevated. Further in vitro protein-binding experiments confirmed that hsp90 increased the binding of CaM to nNOS. Taken together, these data indicate that hsp90 directly augments nNOS catalytic function and that this effect is, at least partially, mediated by CaM-binding enhancement.