Xenobiotics, including drugs such as guanabenz, cause the metabolism-based inactivation of neuronal NO-synthase (nNOS), the covalent alteration of the nNOS heme active site, and proteasomal degradation of the inactivated nNOS. The mechanism of how the inactivated nNOS is selectively culled for degradation is not known. However, we have previously shown that nNOS turnover is regulated by Hsp70/CHIP (C-terminus of Hsp70-interacting protein)-dependent ubiquitination and subsequent proteasomal degradation. In the current study, we show that xenobiotic-inactivated nNOS is selectively ubiquitinated by CHIP in an in vitro system containing purified proteins. Hsp70 facilitates the ubiquitination while Hsp90 protects nNOS from ubiquitination but not from inactivation. Studies in HEK293 cells further support the protective role of Hsp90 on nNOS ubiquitination. A C331A mutant of nNOS, which was previously characterized by Dr. Masters’s lab as having an altered active site conformation, was found to be labilized for Hsp70/CHIP-dependent ubiquitination. The slowly reversible inhibitor, NG-nitro-L-arginine (NNA) protects the C331A nNOS from ubiquitination in in vitro systems as well as in intact cells. The inactive isomer, NG-nitro-D-arginine, did not protect C331A nNOS, further suggesting that the heme active site controls ubiquitination of nNOS. More recently, we found through immunoprecipitation studies that NNA decreases binding of Hsp70 and CHIP to nNOS. Moreover, studies where the oxygenase and reductase domains of nNOS were expressed in HEK293 cells show that Hsp70 and Hsp90 recognize the oxygenase domain. Thus, conformational changes about the heme active site appear to be recognized by chaperones, which then direct the CHIP-dependent ubiquitination. Metabolism-based inactivation and covalent alteration of the heme active site of nNOS is thus a signal for degradation of nNOS and provides a mechanism for surveillance of nNOS protein quality. A more detailed understanding of the nature of the nNOS conformational changes may lead to the design of pharmacological agents, which could alter nNOS levels through changes in rates of degradation of the enzyme. In that clefts are important topological features in nearly all proteins, the ability to detect conformational changes in clefts maybe a general mechanism for surveillance of protein quality by chaperones. Supported by NIH Grants GM077430 and DA022354.