Abstract Effective means to identify the role of reactive oxygen species (ROS) mediating several diseases including cancer, ischemic heart disease, stroke, Alzheimer's and other inflammatory conditions in in vivo models would be useful. The cyclic nitrone 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trap frequently used to detect free radicals in vitro using Electron Paramagnetic Resonance (EPR) spectroscopy. In this study, we synthesized 13C-labeled DMPO for hyperpolarization by dynamic nuclear polarization, in which 13C NMR signal increases more than 10,000-fold. This allows in vivo 13C MRI to investigate the feasibility of in vivo ROS detection by the 13C-MRI. DMPO was 13C-labeled at C5 position, and deuterated to prolong the T 1 relaxation time. The overall yield achieved for 5–13C-DMPO-d 9 was 15%. Hyperpolarized 5–13C-DMPO-d 9 provided a single peak at 76 ppm in the 13C-spectrum, and the T 1 was 60 s in phosphate buffer making it optimal for in vivo 13C MRI. The buffered solution of hyperpolarized 5–13C-DMPO-d 9 was injected into a mouse placed in a 3 T scanner, and 13C-spectra were acquired every 1 s. In vivo studies showed the signal of 5–13C-DMPO-d 9 was detected in the mouse, and the T 1 decay of 13C signal of hyperpolarized 5–13C-DMPO-d 9 was 29 s. 13C-chemical shift imaging revealed that 5–13C-DMPO-d 9 was distributed throughout the body in a minute after the intravenous injection. A strong signal of 5–13C-DMPO-d 9 was detected in heart/lung and kidney, whereas the signal in liver was small compared to other organs. The results indicate hyperpolarized 5–13C-DMPO-d 9 provided sufficient 13C signal to be detected in the mouse in several organs, and can be used to detect ROS in vivo. Graphical abstract fx1 Highlights • Spin traps/radical scavengers cannot be currently imaged by MRI. • 13C labeled spin trap DMPO can be imaged in vivo after hyperpolarization. • Its biodistribution was followed in vivo after hyperpolarization. • 13 C MRI with hyperpolarized DMPO can be used for in vivo free radical detection/imaging. [ABSTRACT FROM AUTHOR]