Abstract —Magnesium (Mg) deficiency enhances tissue sensitivity to ischemic damage, an effect reversed not only by Mg, but also by sulfhydryl (SH)-containing compounds. We therefore created an in vitro model of red blood cell ischemia to investigate whether the protective effects of these compounds might be related to effects on intracellular free Mg (Mg _i ) content. ³¹ P-nuclear magnetic resonance (NMR) spectroscopy was used to measure the high-energy metabolites ATP and 2,3-diphosphoglycerate (DPG) and Mg _i and inorganic phosphate (P _i ) levels in erythrocytes before and for 6 hours after progressive oxygen depletion in the presence or absence of SH-compounds, including captopril, N -acetyl- l -cysteine (NAC), penicillamine, and N -(2-mercaptopropionyl)-glycine (MPG). Under basal aerobic conditions, captopril increased Mg _i in a dose- and time-dependent fashion (174.5±5.3 to 217.1±5.1 μmol/L, P <0.05 at 100 μmol/L, 60 minutes). The SH compounds NAC, penicillamine, and MPG but not the non-SH compound enalaprilat also significantly raised Mg _i in erythrocytes ( P <0.05). With oxygen deprivation, a consistent decrease occurred in both ATP and 2,3-DPG levels associated with a rise in P _i and in the P _i /2,3-DPG ratio used as an index of high-energy metabolite depletion. Captopril, compared with control, retarded the rise in P _i and reduced the P _i /2,3-DPG ratio ( P <0.008 and P <0.025 at 4 and 6 hours, respectively). Furthermore, the higher the initial Mg _i and the greater the captopril-induced rise in Mg _i , the greater the metabolite-protective effect ( r =0.799 and r =0.823, respectively; P <0.01 for both). Altogether, the data suggest that Mg influences the cellular response to ischemia and that the ability of SH compounds such as captopril to ameliorate ischemic injury may at least in part be attributable to the ability of such compounds to increase cytosolic free Mg levels.