Abstract Warburg reported his discovery in Ehrlich ascites carcinoma model, that in the presence of ample oxygen (21% or 150 mmHg O2), cancer cells utilize glucose by “aerobic glycolysis” (Warburg effect), apparently, ascites carcinoma had been assumed to be oxic. However, during past decade, growing evidences indicated that hypoxic cancer cells had evaluated radiolabeled glucose analog18F-FDG uptake than oxic cancer cell in vitro and in vivo. The objective of this study was to observe the oxygen status of ascites fluid and ascites carcinoma in mouse model of ascites carcinoma and its effect on glucose metabolism. Methods: Ascites carcinoma (floating single cells or clusters) and serosa tumors (attached to serosa) were induced by HT29, A549 or MDA-MB-231 cancer cells in the mouse peritoneal cavity. Ascites pO2 was measured using OxyLite systems in ketamine/xylazene anesthetized mice. Hypoxia status in ascites carcinoma and serosa tumors was tested by immunohistochemical visualization of exogenous hypoxia marker pimonidazole binding and HIF1alpha induced glucose transporter-1 expression. Glucose metabolism of was examine by 18F-FDG autoradiography. Effect of carbogen breathing (95% O2 & 5% CO2) on 18F-FDG uptake was investigated. Results: The pO2 of ascites was measured as low as 0.90mmHg (or 0.5% O2) in mice with HT29 ascites carcinomas. In all cell lines we tested, ascites carcinomas was evident 4-6 wk after cell inoculation, all ascites tumors were stained positive for pimonidazole and glucose transporter-1 (GLUT-1), indicating severe hypoxia. Hypoxia status in serosa tumors was heterogeneous and dependent on tumor size. 18F-FDG uptake was significantly higher in ascites and sub-millimeter serosa tumors than in larger ones (generally greater than 1 mm), the former was stained positive for pimonidazole and GLUT-1, but the latter was not. Carbogen breathing (2hrs) significantly reduced 18F-FDG uptake and pimonidazole binding in ascites carcinoma and submillimeter serosa tumors. GLUT1 expression was not affected. Conclusions: In mouse model of ascites carcinoma, ascites and floating ascites carcinoma are severely hypoxic which is in contradict with ample oxygen condition of Ehrlich ascites carcinoma model where “Warburg effect” derived from. Glucose utilization measured by 18F-FDG uptake is oxygen dependent in both ascites carcinoma and serosa tumors, this is unexplained sole by “aerobic glycolysis” of glucose metabolism, thus challenging Warburg effect theory. This study was supported by Kentucky Lung Cancer Research Program Award Citation Format: Xiao-Feng Li. Severe hypoxia drives the increased glucose demand in ascites carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5426. doi:10.1158/1538-7445.AM2013-5426