Although the pseudohalide thiocyanate (SCN-) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN-/H2O2 system and activated eosinophils as roughly equimolar amounts of OSCN- (hypothiocyanite) and OCN- (cyanate). Red blood cells exposed to increasing concentrations of OSCN-/OCN- are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN-/OCN- inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H2O2. Exposure of glutathione S-transferase to [14C]OSCN-/OCN- causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN- /H2O2 reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.