Described here is the application of oxygen isotope fractionation together with computational methods, to elucidate a mechanism of enzymatic H2O2 activation. Horseradish peroxidase (HRP) has been the subject of intensive experimental and computational studies, yet questions remain as to the reversibility of the O-O cleavage step. New insight is afforded by the competitive oxygen kinetic isotope effect (O-18 KIE) upon H2O2 consumption determined under turnover conditions. The 180 KIE is compared to isotope effects calculated for the O-O heterolysis transition state and potential intermediates using density functional theory. In addition, experiments in enriched water provide evidence for HRP-catalyzed scrambling of the O-18 label into the unreacted H2O2. The results provide an unprecedented view of H2O2 activation by a heme peroxidase and challenge the assumption of rate-limiting O-O heterolysis.