Tissue oxygen content is a critical determinant of metabolic functional status. Development of a non-invasive method to quantitatively map (image) tissue oxygen content (pO₂) would be a major advance, enabling assessment of physiologic competence in response to stimulus, disease, and therapeutic intervention. It remains under appreciated that water ¹H longitudinal relaxation in mammalian tissue is well modeled as bi-exponential and it is the experimentally accessible exponential rate constant R_1,slow, characterizing the long-lived relaxation component, that is linearly related to tissue pO₂: R_1,slow = R_1,0,slow + r_1,slow•pO₂. Following appropriate calibration to determine R_1,0,slow and r_1,slow (O₂ relaxivity), an MRI-determined R_1,slow map can, in principle, be converted to a map of tissue pO₂. However, MR-based quantification of tissue pO₂ is challenged by two competing apparent longitudinal relaxation mechanisms: (i) magnetization transfer (MT, characterized by R_1,fast) between the ¹H spins of tissue water and of the solid-like macromolecular matrix (e.g., proteins, cell membranes) and (ii) blood flow, which can bring equilibrium-polarized ¹H spins into the interrogated tissue volume. Herein, we demonstrate that by mitigating the relaxation effects of (i) MT (via discrimination of R_1,slow from R_1,fast) and (ii) blood flow (via non-slice-selective inversion pulses and gradient-enabled IVIM-based vascular spin dephasing), the direct relationship between pO₂ and tissue water longitudinal relaxation can be quantified and tissue pO₂ determined/mapped.•Phantoms. Glutaraldehyde-cross-linked 15% (wt) x-BSA/PBS (bovine serium albumin/phosphate-buffered saline) tissue mimics/phantoms were prepared at various pO₂ by bubbling varying mixtures of O₂ and N₂. Sample pO₂ was measured by an Integra® Licox® Clark-type (platinum) electrode. •In vivo. Normal female BALB/c mice breathed alternate mixtures of 100% O₂, 12.5% O2/87.5% N₂, and 95% O2/5% CO₂ (mixed to modulate tissue pO₂). An OxyLite® optical microprobe (“gold standard”) was implanted into either the right thalamus or thigh muscle to determine tissue-pO₂ in vivo concurrent with MR monitoring. •MRI. Modified Fast Inversion Recovery (MFIR) Point RESolved Spectroscopy (PRESS) data were acquired in x-BSA phantoms and mice: x-BSA - 4.7 tesla; 64 TI from 0.0075 to 6 seconds; non-slice-selective inversion pulse; voxel dimensions = 4x4x4 mm³, two=averages, scan time = 9 min 4 sec; muscle/thalamus in vivo - 1x1x1 mm³ one average, scan time = 4 min 33 sec. •R1 Determination. Relaxation rate constants were estimated using a bi-exponential relaxation model yielding: (i) a “fast” relaxation rate constant (R_1,fast, i.e., MT-dominated relaxation of ¹H spins proximal to the macromolecular matrix, ~20 sec^-1) and (ii) a “slow” relaxation rate constant (R_1,slow, i.e., ¹H spins remote from the macromolecular matrix, ~0.6 sec^-1). Parameter estimates were obtained using Bayesian-probability-theory-based methods: http://bayesiananalysis.wustl.edu/index.html.•x-BSA phantoms. In the x-BSA phantoms, only R_1,slow is sensitive to dissolved O₂ (Figure 1). •In vivo. Employing a blood-flow-mitigating PRESS sequence (non-slice-selective inversion pulse and gradient-enabled IVIM-suppression of vascular ¹H spins) and bi-exponential data modeling, brain and muscle r_1,slow (pO₂ tissue relaxivity in vivo) was determined as 0.56 x 10^-3 ± 0.09 x 10^-3 and 0.69 x 10^-3 ± 0.15 x 10^-3, respectively (summarized in Table inlaid in Figure 2).We report a milestone in the development of a translatable MRI/MRS-based method for quantitatively mapping tissue pO₂. The direct relationship between pO₂ and R₁ (r_1,slow) can be, and has been herein, resolved/quantified by: (i) isolating R_1,slow from MT-dominated R_1,fast and (ii) using non-slice-selective inversion pulses and gradient-enabled IVIM-suppression of vascular ¹H spins) to mitigate blood flow - Quantitative MR Oximetry (QMRO).