Ascorbate free radical is considered to be a substrate for a plasma membrane redox system of eukaryotic cells, and might be involved in stimulation of cell proliferation. It can be generated by transition metal-dependent oxidation of ascorbate or by an equilibrium reaction of ascorbate with dehydroascorbic acid. Using ESR spectroscopic measurements at pH 7.4, we show that when ascorbate and dehydroascorbic acid are mixed at concentrations lower than 2.5 mM, the ascorbate free radical concentration was determined by metal-dependent reactions and not by the equilibrium reaction. We conclude that, for studies under physiological conditions, the ascorbate free radical concentration cannot simply be calculated from the equilibrium constant and the ascorbate and dehydroascorbic acid concentration, but has to be determined experimentally.