As a non-surgical treatment of malignant tumors, it has been experimentally confirmed that the differential hypothermia (D.H.), to keep the tumor normothermic locally under total body hypothermia, showed therapeutic effect on the tumor which was intensified by combination with antitumor agents. The application of D.H. to the treatment of malignant brain tumors in human beings and its mechanism against tumor have been discussed in the previous paper. In order to study the metabolic effect of D.H. on the tumor tissue, the present study was performed by using rabbit Vx2 tumor and applying D.H. for 10hours. The concentrations of major metabolites of the glycolytic pathway (glucose, G-6-P, F-6-P, FDP DAP, pyruvate, lactate) and high energy phosphate bonds (ATP, ADP, AMP, P-creatine) were measured by enzymatic techniques before, during and after the D.H. treatment. 1) The G-6-P concentration in D.H.-treated tumors showed an elevation as compared with that in control. Its concentration rose to 0.251 ± 0.044 mol/g immediately after the D.H. treatment and to 0.233 + 0.030 mol/g at 36th hour later. 2) Lactate concentration in D.H.-treated tumors also showed an elevation. Its concentration at 6th hour following the D.H. treatment was 18.6 ± 1.0 mol/g at 36th hour it was 19.0 ± 1.8 mol/g. Lactate/pyruvate ratio also elevated. 3) ATP concentration in D.H.-treated tumors decreased. Its concentration at 6th hour during the D.H. treatment was 0.708 ± 0.108 mol/g and at 36th hour it was 0.428 ± 0.090 mol/g. D.H.-treated tumors revealed a decrease of energy charge potentials. These experimental results are considered to show a metabolic mechanism of the antitumor effect of the D.H. treatment and seem to be compatible with the morphological evidence of tumor disappearance reported in the previous paper.