Tissue hypoxia is a prevalent physiological condition in various pathologies such as cancer, ischemic heart disease and stroke [¹]. Rapid proliferation of cancer cells combined with unbalanced levels of pro and anti-angiogenic factors lead to abnormal vasculature which results in regions of hypoxia in solid tumors [^2,3]. The increase in hypoxic regions and stress causes metabolic changes in the tumors, giving rise to abnormal pathways promoting metastasis, proliferation, resistance to chemo and radiotherapy and poor prognosis. Targeted, non-invasive in vivo imaging of hypoxia has the potential to determine regions with poor oxygenation and differentiate between normoxic vs hypoxic tissues. MRI provides a powerful platform for generating three-dimensional spatial maps of hypoxia with the use of a novel hypoxia binding contrast agent which could immediately impact the therapeutic choices. In the present study, we report in-vitro and in-vivo analysis of a hypoxia targeted Gadolinium (Gd³⁺) based MRI contrast agent at 7T. GdDOTA monoamide conjugate of 2-nitroimidazole, GdDO3NI was prepared as previously described [^4,5] and relaxivity studies were conducted on a Bruker Biospec 7T system at bore temperature (~23.6°C) and 37°C using serial dilution DI phantom (0-2mM). In-vivo study was carried out on nu\nu mice implanted subcutaneously with NCI-H1975 (NSCLC) or A431 (epidermoid carcinoma) tumor cell line in the right thigh. 1 mm thick T1-weighted images were acquired pre and post injection of 0.3 mmole\kg body weight contrast agent for every 5 minutes up to 120 minutes post injection to a total time of 150 minutes. Pimonidazole was used as an ex-vivo marker for hypoxia and tumors were harvested for IHC staining. MR percentage enhancement images were co-registered with IHC images in Matlab. R² values between the tumor boundaries and hypoxia boundaries were then determined using custom Matlab code to correlate the degree of correlation for the images.A linear fit to the relaxation rates (R1 & R2) vs concentration data yielded the reflexivity r1 and r2 value of 4.75 ± 0.04 mM^-1 s^-1 and 7.52 ± 0.07 mM^-1 s^-1, respectively. Three tumors were imaged with MRI and immunohistochemically. When registered, the tumor boundaries yielded R² values of 0.9034, 0.9347, and 0.9066 for tumor 1 slice 1, tumor 1 slice 2 and tumor 2 respectively on figure 2. Tumor 1 slice 1 and slice 2 are of an H-1975 cancer cell line, and tumor 2 an A431 cell line. The hypoxia-thresholded R² values yielded 0.4752, 0.3057, and 0.4100 for tumor 1 slice 1, tumor 1 slice 2 and tumor 2 respectively. The corresponding hypoxic fractions and comparisons between MRI and IHC are listed in figure 1. The goal of this study was to establish GdDO3NI as a potential hypoxia mapping contrast agent that would aid in studying the physiological properties on tumors in individual patients and hence help direct the treatment decisions and study the tumor’s response to treatments. Immunohistochemical studies correlate with the contrast enhancement maps obtained and showcase the agent’s ability to effectively report regions of hypoxia. Figure 2 provides a visual for the hypoxia binding, with each tumor showing hypoxia in the same general areas between the two modalities. The R² values for hypoxia thresholded maps are smaller than expected, however. This is hypothesized to be a result of the difference in slice thickness between the modalities (1mm for MRI, 20 µm for IHC, a factor of 50) as well as any local mismatches in co-registration. This leads to some discrepancies in tissue content between the two modalities, leading to reduction in voxel-by-voxel match between the two. The bulk hypoxic fractions also were close in value between the modalities, with the exception of tumor 1 slice 2. Future work will involve the collection of several of these IHC images to reconstruct a 1mm thick IHC image to more accurately correlate with the two as well as on 3-dimensional tumor spheroids in vitro.