Recently, a T2-Relaxation-Under-Spin-Tagging (TRUST) MRI technique was developed to quantitatively estimate blood oxygen saturation fraction (Y) via the measurement of pure blood T2. This technique has shown promise for normalization of fMRI signals, for the assessment of oxygen metabolism, and in studies of cognitive aging and multiple sclerosis. However, a human validation study has not been conducted. In addition, the calibration curve used to convert blood T2 to Y has not accounted for the effects of hematocrit (Hct). In this study, we first conducted experiments on blood samples under physiologic conditions, and the Carr-Purcell-Meiboom-Gill T2 was determined for a range of Y and Hct values. The data were fitted to a two-compartment exchange model to allow the characterization of a three-dimensional plot that can serve to calibrate the in vivo data. Next, in a validation study in humans, we showed that arterial Y estimated using TRUST MRI was 0.837 ± 0.036 (N=7) during the inhalation of 14% O2, which was in excellent agreement with the gold-standard Y values of 0.840 ± 0.036 based on Pulse-Oximetry. These data suggest that the availability of this calibration plot should enhance the applicability of T2-Relaxation-Under-Spin-Tagging MRI for noninvasive assessment of cerebral blood oxygenation. Magn Reson Med, 2011. © 2011 Wiley-Liss, Inc.