The Fe 2+/vitamin C system has often been used to initiate in vitro lipid peroxidation. In this paper, we attempt to locate the optimal conditions of the Fe 2+/vitamin C system to be used in lipid peroxidation experiments. We have measured the quantity of lipid peroxidation through assessment of thiobarbituric acid reactive substances. We tested the influence of the Fe 2+ and vitamin C concentration, the pH, the reaction temperature, and different buffer systems on the extent of lipid peroxidation in preparations of pig heart membranes. The optimum rate of lipid peroxidation occurred at an Fe 2+/vitamin C ratio of 1:5. Decreasing pH and changing the buffer system resulted in changes of the extent of peroxidation. We also show that the EPR spin trap technique with 5,5-dimethyl-pyrroline-1-oxide (DMPO), which is used to detect free radicals in the Fe 2+/vitamin C system, cannot work. This is because vitamin C immediately quenches the formed DMPO adducts by reduction. In addition, no evidence was found as to the nature of Fe 2+/vitamin C formed radicals in experiments using superoxide dismutase, catalase or mannitol, butylhydroxyanisol, and α-tocopherole as radical scavengers.