Abstract Hypoxia, a condition of oxygen deprivation, is a salient feature of the tumor microenvironment. Hypoxia tumors are frequently resistant to conventional cancer therapies and are correlated with increased migratory and metastatic behavior. A number of detection modalities exist for the detection of hypoxia; however, only EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide) provides detailed information on the distribution of hypoxia in tissues at both the physiologic and pathologic levels. EF5 is a compound that, when injected into animal tissues, selectively binds to hypoxic cells and forms adducts. These adducts are detected by a mouse monoclonal antibody, clone ELK3-51, conjugated to fluorescent dyes including Cyanine 3, Alexa Fluor 488 and Cyanine 5. The use of EF5 and its specific fluorescent monoclonal antibody to selectively detect regions of hypoxia in mouse tissues is described. BALB/c mice between 7 to 10 weeks old and weighing between 18 to 30 g were used in the study. Mouse #1 was not treated with EF5 and its spleen was harvested as a negative control. Mouse #2 was injected with 10 mM EF5 (typically 1% of animal weight in mL) and allowed to run aerobically for 4 hours before euthanization. Mouse #3 was injected with EF5, euthanized after 10 minutes and kept warm at 37°C for 40 minutes to mimic the anaerobic hypoxic condition before the spleen was harvested (hypoxic control). Examination of spleen samples indicated highly specific binding of the EF5 adducts only in the anaerobic hypoxic mouse spleen. The use of EF5 with fluorescent conjugated antibodies provides a sensitive and quantitative method for the detection and measurement of tissue hypoxia in animal and human tumor tissues and cells. Citation Format: Kevin Su, Amy Noble, Nick Asbrock, Cameron Koch, Vi Chu. Detection of tissue hypoxia with EF5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2442.