The in vitro oxidation of tryptophan (Trp) by pro-oxidant systems such as iron-ascorbate indicates that Trp is a target for oxygen radicals in vivo. The Trp in albumin and lipoproteins has been reported to be actively oxidized by hydroxyl radical (HO(•)) generating systems such as copper-ascorbate or PUFA (polyunsaturated fatty acids) respectively. The super-physiological concentrations of the oxidants used in these studies prompted us to examine the effect of low copper and ascorbate concentrations on Trp oxidation. Trp (10-5000 μmol/L) was incubated with 1.5 μmol/L copper plus ascorbate (0.113 and 1.13 mmol/L) at 37°C and its oxidation followed by fluorescence and high-performance liquid chromatography. The percentage of Trp oxidized by the ascorbate-copper system was inversely related to its concentration and positively related to the ascorbate concentration. High concentrations of Trp (above 50 μmol/L for 0.113 mmol/L and 500 μmol/L for 1.13 mmol/L ascorbate) are not significantly oxidized in the presence of ascorbate. The large drop in the percentage Trp oxidation at higher concentrations may be due to the chelation of copper by Trp. High concentrations of Trp (over 50 μmol/L) strongly prevented ascorbate oxidation by copper, and therefore inhibited the production of HO(•) needed for Trp oxidation. Protein Trp is less readily oxidized by the ascorbate-copper system than free Trp. Proteins chelate copper much better than Trp, and so inhibit its oxidative activity, at least against ascorbic acid.