In hypoxic tumor microenvironments, the strongly reducing redox environment reduces evofosfamide (TH-302) to release a cytotoxic bromo-isophosphoramide (Br-IPM) moiety. This drug therefore preferentially attacks hypoxic regions in tumors where other standard anticancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO . Here, we use quantitative pO imaging by electron paramagnetic resonance (EPR) to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinoma xenograft models: MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors that do not respond. EPR imaging showed that oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in K observed in dynamic contrast-enhanced magnetic resonance imaging (DCE MRI). The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and antiproliferatives post evofosfamide for improved outcomes. This study suggests that reoxygenation after evofosfamide treatment is due to decreased oxygen demand rather than improved perfusion. Following the change in pO after treatment may therefore yield a way of monitoring treatment response. . 35, 904-915.