The promising dynamic nuclear polarization (DNP) for hyperpolarized 13C-MRI/MRS of real-time metabolism in vivo is challenged by the limited number of agents with the required physical and biological properties. The physical requirement of a liquid-state T1 of tens of seconds is mostly found for 13C-carbons in small molecules that have no direct protons attached, i.e. carbonyl, carboxyl and certain quaternary carbons. Unfortunately, such carbon positions do not exist in a large number of metabolic agents, and chemical shift dispersion often limits detection of their chemical evolution. We have previously shown that direct deuteration of protonated carbon positions significantly prolongs the 13C T1 in the liquid state and provides potential 13C-labeled agents with differential chemical shift with respect to metabolism. The Choline Molecular Probe [1,1,2,2-D4, 2-13C]choline chloride (CMP2) has recently been introduced as a means of studying choline metabolism in a hyperpolarized state. Here, the biophysical properties of CMP2 were characterized and compared with those of [1-13C]pyruvate to evaluate the impact of molecular probe deuteration. The CMP2 solid-state polarization build-up time constant (30 min) and polarization level (24%) were comparable to those of [1-13C]pyruvate. Both compounds' liquid state T1 increased with temperature. The high-field T1 of CMP2 compared favorably with [1-13C]pyruvate. Thus, a deuterated agent demonstrated physical properties comparable to a hyperpolarized compound of already proven value, whereas both showed chemical shift dispersion that allowed monitoring of their metabolism. It is expected that the use of deuterated carbon-13 positions as reporting hyperpolarized nuclei will substantially expand the library of agents for DNP-MR. Copyright © 2011 John Wiley & Sons, Ltd.