Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the ubiquitous protein chaperone hsp90. We have shown previously that nNOS expressed in Sf9 cells where endogenous heme levels are low is activated from the apo- to the holo-enzyme by addition of exogenous heme to the culture medium, and this activation is inhibited by radicicol, a specific inhibitor of hsp90 (Billecke, S. S., Bender, A. T., Kanelakis, K. C., Murphy, P. J. M., Lowe, E. R., Kamada, Y., Pratt, W. B., and Osawa, Y. (2002) J. Biol. Chem. 278, 15465-15468). In this work, we examine heme binding by apo-nNOS to form the active enzyme in a cell-free system. We show that cytosol from Sf9 cells facilitates heme-dependent apo-nNOS activation by promoting functional heme insertion into the enzyme. Sf9 cytosol also converts the glucocorticoid receptor (GR) to a state where the hydrophobic ligand binding cleft is open to access by steroid. Both cell-free heme activation of purified nNOS and activation of steroid binding activity of the immunopurified GR are inhibited by radicicol treatment of Sf9 cells prior to cytosol preparation, and addition of purified hsp90 to cytosol partially overcomes this inhibition. Although there is an hsp90-dependent machinery in Sf9 cytosol that facilitates heme binding by apo-nNOS, it is clearly different from the machinery that facilitates steroid binding by the GR. hsp90 regulation of apo-nNOS heme activation is very dynamic and requires higher concentrations of radicicol for its inhibition, whereas GR steroid binding is determined by assembly of stable GR.hsp90 heterocomplexes that are formed by a purified five-chaperone machinery that does not activate apo-nNOS.