Among the biologically and pharmacologically relevant nitrogen oxides, nitroxyl (HNO) remains one of the most poorly studied and least understood. Several previous reports indicate that thiols may be a primary target for the biological actions of HNO. However, the intimate details of the chemical interaction of HNO with biological thiols remain unestablished. Due to their ability to grow under a variety of conditions, the yeast Saccharomyces cerevisiae represents a unique and useful model system for examining the chemistry of HNO with thiol proteins in a whole-cell preparation. Herein, we have examined the effect of HNO on the thiol-containing, metal-responsive, yeast transcription factor Ace1 under a variety of cellular conditions as a means of delineating the chemistry of HNO interactions with this representative thiol protein. Using a reporter gene system, we find that HNO efficiently inhibits copper-dependent Ace1 activity. Moreover, this inhibition appears to be a result of a direct interaction between Ace1 thiols and HNO and not a result of any chemistry associated with HNO-derived species. Thus, this report indicates that thiol proteins can be a primary target of HNO biochemistry and that HNO-mediated thiol modification is likely due to a direct reaction of HNO.