The metabolism-based inactivation of neuronal NO-synthase (nNOS) leads to 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. We have previously shown that nNOS turnover is regulated by Hsp70/CHIP (C-terminus of Hsp70-interacting protein)-dependent ubiquitination. We now 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. A C331A mutant of nNOS, which was previously characterized by Dr. Masters’s lab as having an altered active site conformation, is labilized for Hsp70/CHIP-dependent ubiquitination. The slowly reversible inhibitor, NG-nitro-l-arginine (NNA), but not the D-isomer of NNA, protects the C331A and wild type nNOS from ubiquitination in in vitro systems as well as in intact cells. More recently, we found that NNA decreases binding of Hsp70 and CHIP to nNOS. 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. In that clefts are important topological features in nearly all proteins, the ability to detect these conformational changes maybe a mechanism for maintaining nNOS protein quality through chaperones, as well as play an important role in the chaperone-facilitated insertion of heme into the active site cleft of apo-nNOS (Supported by NIH GM077430).