The effects of phosphate (Pi) and ascorbic acid on Fe(II)-induced lipid peroxidation were studied in rat liver mitochondria (RLM) at 25℃ The examination was conducted with RLM at a concentration of 0.5mg protein/ml, 0.05mM Fe(II), 2mM Pi and 0.5mM ascorbic acid. Peroxidation was detected by the thiobarbituric acid reaction (TBA-value), and the concentration of Fe (II) in the reaction mixture was determined simultaneously by the Nitroso-PSAP method. Fe(II)-induced peroxidation had a lag period followed by a burst in the TBA-value and then held a moderate level. A slow decrease in Fe(II) content was observed during the lag period which was followed by a greater decrease that paralleled the burst in the TBA-value. The Fe(II) concentration at the initiation of the burst was one-third of the added Fe(II) concentration. The ascorbic acid-induced peroxidation had a lag period accompanied by a slow increase in the TBA-value. The Fe(II)•ascorbic acid-induced peroxidation had a longer lag period than the ascorbic acid-induced one. The TBA-value increased more slowly, and the Fe(II) concentration was not reduced in this case. Peroxidation induced by Fe(II)•Pi had a shorter lag period and a greater TBA value than that by Fe(II). The Fe(II) content was reduced more rapidly with Fe(II)•Pi than with Fe(II) only. Ascorbic acid•Pi-induced peroxidation showed an elongated lag period compared with ascorbic acid only, and the TBA-value increased more slowly. Fe(II)•Pi•ascorbic acid-induced peroxidation had a slightly longer lag period than the peroxidation induced by Fe(II)•Pi, and had the highest TBA-value. Fe(II) content was reduced during the lag period, and then increased. Other experiments under this condition indicated that the rate of Fe(II) content reached the optimum level needed for the initiation of burst peroxidation.