The effect of differential hypothermia (D.H.) combined with anti-tumor agent, 5-fluorouracil (5-FU), was described in the previous report (Part 1). In this study, the tissue levels of 5-FU in the tumor and other organs during D.H. treatment were examined by bioassay method. At first, using Vx2 tumors transplanted into the bilateral ears of the rabbits, D.H. treatment was performed with the same procedures as Part 1. The tissue level of 5-FU in the tumors, detected 45 minutes after administration, was the highest in the D.H.-treated tumors (22.1μg/g), as compared with contra-lateral, hypothermic tumors (11.4μg/g) and non-treated, normothermic tumors (6.8μg/g). The tissue level of 5-FU in other organs such as lung, kidney, skin, liver and brain was low in both hypothermic and normothermic conditions. But blood level of 5-FU was kept relatively high during generalized hypothermia (1hr.; 150μg/ml, 2hrs.; 10.4μg/ml), as compared with normothermia (1hr.; 5.5μg/ml, 2hrs.; 0.5μg/ml). The author further investigated that the tissue level of 5-FU in the brain tumors (Vx2 tumors transplanted into the brain of rabbit) was affected with D.H. treatment. In this experiment, the tumors were kept normothermic by irradiation of 2.45 GHz microwave under general body coiling. The D.H. treated brain tumors revealed higher level of 5-FU (19.6μg/g) than the nontreated brain tumors (6.4μg/g).