Purpose: To develop a magnetic resonance (MR) imaging approach to noninvasively image quantitative Po2 in the human vitreous. Materials and Methods: Human studies were approved by the institutional review board with informed consent obtained from all subjects and were HIPAA compliant. Animal studies were performed with animal care committee approval. An MR imaging method to measure the longitudinal relaxation rate, or R1, of water was implemented with a 3.0-T MR imager. R1 was calibrated in water phantoms at multiple Po 2 and temperature conditions (n = 10) and in ex vivo animal vitreous (n = 2). Vitreous Po2 was imaged in three human volunteers (age range, 26-28 years) in multiple sessions on separate days to evaluate reproducibility. The effects of temperature and ambient air were evaluated by acquiring data with the eye open and closed. Statistical analysis consisted of t tests, with P less than .05 indicating significant difference. Results: Calibrations of phantoms and ex vivo vitreous yielded an R1 association with oxygen of 0.209 sec-1 + Po2 · 2.07 × 10 -4 sec21/mm Hg at 37°C, and an association with temperature (Δ[1/R1]/ΔTemperature) of 0.106 sec/°C ± 0.009 (standard deviation). A difference in R1 was found between the phantoms and vitreous. If uncorrected, vitreal Po2 would be significantly overestimated (P < .001). In vivo human vitreous Po2 maps were spatially heterogeneous, with a whole vitreous Po2 of 16.7 mm Hg ± 6.5 (eye closed). Measurements between open and closed eyes showed spatially dependent R1 differences, which translated to temperature differences of 0.34°-0.83°C across the eye. Conclusion: This study established an MR imaging protocol to image quantitative vitreous Po2 noninvasively and evaluated effects from vitreal macromolecules, temperature gradients, and ambient air on vitreal Po2 values. Measurement of vitreous Po2 with MR imaging has the potential to be used to study eye diseases noninvasively.