Ascorbate releases iron from the ferritin core by reducing ferric to soluble ferrous iron. A novel finding is that labile iron > 2 μM synergises ascorbate-dependent iron mobilization by decreasing the Km ( 1.5 mM) and increasing Vmax; both by approximately five-fold, (P<0.001). Hydrogen peroxide act as oxidizing agent on core iron and competitively inhibits ascorbate’s action there. Labile free iron reverses these effects.Iron mobilization by ascorbate alone has a higher activation energy (Ea = 45±5.5 S.E.M. kJ/mole) than with both ascorbate and labile iron (Ea = 13.7±2.2 S.E.M. kJ/mole P< 0.01). Ascorbate alone mobilization has a 3-fold lesser pH sensitivity (pH range 6.0-8.0) than the combination of ascorbate and iron (P< 0.01). Ascorbate and hydrogen peroxide compete for the bi-ferrous/ferric ferroxidase centre within the ferritin core and L-012 chemiluminescence demonstrates that hydroxyl radical is displaced from ferritin by catalase-sensitive action of ascorbate alone (50-250 μM). Superoxide radical generated by ferritin, as monitored by the chemiluminescence probe Lucigenin, is increased by the simultaneous presence of ascorbate and labile iron. This superoxide signal is enhanced by catalase and abolished by catalase and SOD acting together. Ascorbate alone has no effect on ferritin’s Lucigenin chemiluminescence. These chemiluminescence studies demonstrate a dual mechanism of ascorbate-dependent mobilization. These results are consistent with the view that labile iron changes the mechanism of ascorbate’s reduction of ferritin iron from a simultaneous two electron process to a one electron transfer process catalysed by several possible one electron donors e.g. superoxide, ascorbyl radical, ferryl radical and quercetin semiquinone radical. These findings have several implications relating to inflammatory disorders, since labile iron synergises ascorbate-dependent iron release from ferritin in the normal physiological concentration range hence explaining ascorbate’s pro-oxidant effects at physiological concentrations (50-250 μM).