Nitroxyl (HNO) was found to inhibit glycolysis in the yeast Saccharomyces cerevisiae. The toxicity of HNO in yeast positively correlated with the dependence of yeast on glycolysis for cellular energy. HNO was found to potently inhibit the crucial glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an effect which is likely to be responsible for the observed inhibition of glycolysis in whole cell preparations. It is proposed that GAPDH inhibition occurs through reaction of HNO with the active site thiolate residue of GAPDH. Significantly, levels of HNO that inhibit GAPDH do not alter the levels or redox status of intracellular glutathione (GSH), indicating that HNO has thiol selectivity. The ability of HNO to inhibit GAPDH in an intracellular environment that contains relatively large concentrations of GSH is an important aspect of HNO pharmacology and possibly, physiology.