Ferrous ion-induced lipid peroxidation of the mitochondria isolated from rat liver on the 3rd day after x-ray whole-body irradiation at 650R showed shortening of the lag of induction period and more acceleration of the activity than those of normal mitochondria (Part 1). Further investigations were made on the mitochondria on the 3rd day after irradiation at 650R in regard to fatty acid compositions and Fe(++) -induced peroxidation of total or fractionated mitochondrial lipids. The results are represented as follows. 1) Fatty acid composition of the mitochondria after lipid peroxidation showed the decrease of polyenoic acids (C-20:4, C-22:6), suggesting the polyenoic acids are substrate of the reaction. 2) Changes of fatty acid composition of mitochondria after whole-body irradiation at 650R were shown decreasing of unsaturated fatty acid due to the decrement of C-18:1 and C-18:2, but the component of polyenoic acid increased relatively. These changes are transient, reaching a maximum on the 3rd day after irradiation, and this tendency is parallel to that of lipid peroxidation activity of the mitochondria whole-body irradiated. 3) No difference of the rate of peroxidation observed between total lipids extracted from normal and from whole-body irradiated mitochondria, and the lag of induction period was not seen in both reactions. 4) Peroxidation of the total lipid was seen markedly in the phospholipid fraction and slightly in the simple lipid fractions. Effect of whole-body irradiation on the peroxidation activities of the phospholipid was not observed significantly, despite of the difference seen in their fatty acid compositions. 5) Peroxidation of subfractionated phospholipid by a thin-layer chromatography showed marked activity in the fractions of lecithin and aminophosphatide containing large amounts of C-20:4 and C-22:6: Recovery of the activity of the subfractions increased markedly comparing to the total phospholipid, and effect of whole-body irradiation appeared significantly in these subfractions. However, relationship between activities of the peroxidation and fatty acid compositions of the subfractions cannot seen. It is suggested the mutual interaction of phopholipid subfractions on the peroxidation to decrease the activity, and some of lipid became more sensitive to peroxidation by irradiation. 6) Relative amount of phospholipid to total lipid increased in whole-body irradiated samples. 7) From these findings, it was discussed that the acceleration of Fe(++) -induced lipid peroxidation in mitochondrial level is due to the change of fatty acid composition and association of lipid in the membrane.