Nitroxyl (HNO) has received recent and significant interest due to its novel and potentially important pharmacology. However, the chemical/biochemical mechanism(s) responsible for its biological activity remain to be established. Some of the most important biological targets for HNO are thiols and thiol proteins. Consistent with this, it was recently reported that HNO inhibits the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein with a catalytically important cysteine thiol at its active site. Interestingly, it was reported that intracellular GAPDH inhibition occurred without significantly altering the cellular thiol redox status of glutathione. Herein, the nature of this reaction specificity was examined. HNO is found to irreversibly inhibit GAPDH in a manner that can be protected against by one of its substrates, glyceraldehyde-3-phosphate (G-3-P). These results are consistent with the idea that HNO has the ability to react with and oxidize a variety of intracellular thiols and the ease or facility of cellular re-reduction of the thiol targets can determine the target specificity.