NAD-linked α-glycerophosphate dehydrcgenase (G3P oxidoreductase, EC 1. 1. 1. 8: G3P: NAD 2-oxidoreductase) and FAD-linked α-glycerophosphate dehydrogenase (EC I.I.99.5:G3P: cytochrome oxidoreductase) of sarcoma 180 ascites tumor cells in mice were studied. The activity of NAD-linked α-glycerophosphate dehydrogenase activity was very low in cytosolic fraction of tumor cells. It was shown that this amzyme activity reduced by one-hundredth as compared with that of normal mouse liver. FAD-linked α-glycerophosphate dehydrogenase activity in mitochondria) fraction was found to be remarkable low. The level of this enzyme was about one-third when compared to that in cytosolic NAD-linked enzyme. The activity of cytosolic NAD-linked α-glycerophosphate dehydrogenase was fo und to increase slightly in 7-day-old tumor cells, and thereafter declined to initial value in 14-day-old tumor cells. Mitochondrial FAD-linked α-glycerophosphate dehydrogenase activity was almost unchanged during first 1 week, then increased to about twice the initial value in 14-day-old tumor cells. Isozymes of cytosolic fraction of tumor cells and ascites fluid were studies. Samples were precipitated by 30 per cent ammonium sulfate saturation, followed by 70 per cent saturation. The precipitate was dialysed and subjected to Sephadex G-100 and DEAE-cellulose column chromatography under linear concentration gradient elution. The effluents from 3-, 7-, and 14-day-old ascites fluid samples contained two peaks, respectively, which exhibited NAD-linked α-glycerophosphate dehydrogenase activity. The activity of these isozymes was found to increase during the tumor growth. However, NAD-linked α-glycerophosphate dehydrogenuse in cytosolic fraction of tumor cells showed no multiple molecular forms throughout the developmental period of 2 weeks. Cytosolic NAD-linked α-glycerophosphate dehydrogenase has been further 910 fold purlified by affinity chromatography and some of its molecular properties were studied. The molecular weight was determined to be approximately 62,000 by sodium dodecylsulfate polyacrylamide disc electrphoresis. The apparent Km of cytosolic α-glycerophosphate dehydrogenase at pH 9.0 is 0.83 mM for α-glycerophosphate and 2.0 mM for NAD, respectively. The pH optimum for the enzyme is 9.0 The potimal temperature is about 55℃. The enzyme was activated by bivalent actions. The sulfhydryl inhibitor, p-chloromercuribenzoate, profoundly reduced enzymatic activity.