Quantitative in vivo oximetry has been reported using 19F MRI in conjunction with reporter molecules, such as perfluorocarbons, for tissue oxygenation (pO2). Recently, hexamethyldisiloxane (HMDSO) has been proposed as a promising alternative reporter molecule for 1H MRI-based measurement of pO2. To aid biocompatibility for potential systemic administration, we prepared various nanoemulsion formulations using a wide range of HMDSO volume fractions and HMDSO to surfactant ratios. Calibration curves (R1 versus pO2) for all emulsion formulations were found to be linear and similar to neat HMDSO for low surfactant concentrations (< 10% v/v). A small temperature dependence in the calibration curves was observed, similar to previous reports on neat HMDSO, and was characterized to be approximately 1 Torr/ °C under hypoxic conditions. To demonstrate application in vivo, 100 µL of this nanoemulsion was administered to healthy rat thigh muscle (Fisher 344, n = 6). Dynamic changes in mean thigh tissue pO2 were measured using the PISTOL (proton imaging of siloxanes to map tissue oxygenation levels) technique in response to oxygen challenge. Changing the inhaled gas to oxygen for 30 min increased the mean pO2 significantly (p < 0.001) from 39 ± 7 to 275 ± 27 Torr. When the breathing gas was switched back to air, the tissue pO2 decreased to a mean value of 45 ± 6 Torr, not significantly different from baseline (p > 0.05), in 25 min. A first-order exponential fit to this part of the pO2 data (i.e. after oxygen challenge) yielded an oxygen consumption-related kinetic parameter k = 0.21 ± 0.04 min−1. These results demonstrate the feasibility of using HMDSO nanoemulsions as nanoprobes of pO2 and their utility to assess oxygen dynamics in vivo, further developing quantitative 1H MRI oximetry. Copyright © 2011 John Wiley & Sons, Ltd.