HPLC/electrochemical detection was used to identify five major low MW water soluble electrochemically active molecules from the aqueous humor of three species of mammals: New Zealand White rabbits and humans (diurnal) and Sprague-Dawley rats (nocturnal). These molecules are L-cysteine (CYS), L-ascorbic acid (AA), glutathione (GSH), uric acid (UA) and L-tyrosine (TYR); all of these molecules have known antioxidant properties. Nocturnal rat aqueous humor is concentrated in two thiols: GSH (125 microM; n = 24 pooled eyes) and CYS (63 microM), in contradistinction to diurnal species which have high concentrations of AA. No deterioration of any of these antioxidants occurs in a synthetic aqueous humor mixture irradiated with a physiologically relevant spectral UV B dose of 30 mJ/cm2/h (5.5 UV equivalent sunlight hours). The same result occurred with addition of the endogenous aqueous humor UV B photosensitizer L-tryptophan. In a second set of experiments, human synthetic aqueous humor was subjected to hydrogen peroxide induced oxidant stress. The decay of antioxidants was CYS > GSH > AA > UA > TYR. The second highest concentrated antioxidant in human aqueous humor is TYR. Yet TYR failed to protect AA against H2O2-induced free radical damage in a synthetic aqueous humor model system (P = 0.10; ANOVA). The existence of multiple electrochemically active constituents and their thermodynamic interactions must be recognized when choosing animal models to evaluate human aqueous humor antioxidant defense.