The influence of vitamins E and C on the initial stages of lipid peroxidation in human erythrocyte membranes was assessed with the fluorescent polyunsaturated fatty acid, parinaric acid, as probe molecule. Cumene hydroperoxide was used as initiator with either haemin-Fe 3รท or Cu 2. as metal ion cofactor. The effect of vitamin C (pro-or antioxidant) appeared to be determined by the localisation of the metal ions, either in the water phase or in the membrane. Vitamin C is only able to reduce metal ions in the water phase, which results in acceleration of radical generation and subsequent enhancement of parinaric acid peroxidation. Thus, interaction of vitamin C with Cu 2รท in the water phase led to drastically enhanced peroxidation of parinaric acid. In contrast, when only membrane-associated haemin-Fe ~ยฐ was present, vitamin C functioned as an antioxidant at all concentl"ations tested (0--10 ~M). In a system with haemin-Fe 3ยฐ equilibrated between the water phase and the membranes, < 5 0M vitamin C produced an overall prooxidant, and > 15/~M vitamin C an overall antioxidant effect. At vitamin C concentrations of 5--15/aM, continuous measurement of parinaric acid fluorescence revealed a shift in the vitamin C effect from antioxidant to prooxidant within the time-course of an assay. Vitamin E exhibited a protective effect on peroxidation initiated by cumene (per)oxyl radicals with haemin-Fe 3รท as cofactor, by inducing a concentration-dependent extension of the lag-phase in parinaric acid peroxidation. Vitamin E appeared to be much more effective compared with vitamin C in scavenging radicals in this system. This indicates that vitamin C has only a limited ability to react with cumene (per)oxyl radicals in the membrane. The combination of vitamins E and C produced a protective effect on parinaric acid peroxidation exceeding the sum of their individual contributions. Moreover, the rate of vitamin E consumption was drastically lowered in the presence of vitamin C, whereas the rate of vitamin C consumption hardly decreased in the presence of vitamin E. The results are discussed in terms of a reaction scheme where the relative contributions of a number of reactions are considered to determine the total effect of added vitamin C or E. Vitamin E radicals constitute an additional substrate for vitamin C, resulting in a more than additive shift in the overall effect to the antioxidant side.