Toxicity mediated by free heme has emerged as an important element of end organ injuries and adverse outcomes in critically ill disease states. Free heme is thought to be derived from oxidative denaturation of free hemoglobin, secondary to red cell hemolysis. In this study, we evaluated the ability of oxidants (H2O2, nitrite, peroxynitrite and hypochlorous acid) formed during inflammation to cause heme release from purified hemoglobin and hemolysates, at pH 7.4 and 6.8. Supraphysiological concentrations of nitrite, peroxynitrite or hypochlorous acid were required to cause appreciable heme release from either free hemoglobin or hemolysates. However, H2O2 administered as a bolus or generated in situ, was more potent at promoting free heme release with free hemoglobin. With hemolysates, only in situ H2O2 formation resulted in significant free heme release. In all cases, free heme release was higher at lower pH and required oxidation of ferrous heme, but was not dependent on ferrylHb formation. Moreover, ligating ferric heme with cyanide or blocking the β93Cys did not prevent, but in fact increased free heme release. The salient observations from this study are that free heme release is likely mediated by continuous generation of H2O2 versus other heme oxidants, and facilitated at low pH.