Choline as a reporter molecule has been investigated by in vivo magnetic resonance for almost three decades. Accumulation of choline metabolites (mainly the phosphorylated forms) had been observed in malignancy in preclinical models, ex-vivo, in vivo and in patients. The combined choline metabolite signal appears in 1H-MRS of the brain and its relative intensity had been used as a diagnostic factor in various conditions. The advent of spin hyperpolarization methods for in vivo use has raised interest in the ability to follow the physiological metabolism of choline into acetylcholine in the brain. Here we present a stable-isotope labeled choline analog, [1,1,2,2-D4,2-13C]choline chloride, that is suitable for this purpose. In this analog, the 13C position showed 24% polarization in the liquid state, following DNP hyperpolarization. This nucleus also showed a long T1 (35 s) at 11.8 T and 25 °C, which is a prerequisite for hyperpolarized studies. The chemical shift of this 13C position differentiates choline and acetylcholine from each other and from the other water-soluble choline metabolites, namely phosphocholine and betaine. Enzymatic studies using an acetyltransferase enzyme showed the synthesis of the deuterated-acetylcholine form at thermal equilibrium conditions and in a hyperpolarized state. Analysis using a comprehensive model showed that the T1 of the formed hyperpolarized [1,1,2,2-D4,2-13C]acetylcholine was 34 s at 14.1 T and 37 °C. We conclude that [1,1,2,2-D4,2-13C]choline chloride is a promising new molecular probe for hyperpolarized metabolic studies and discuss the factors related to its possible use in vivo. Copyright © 2010 John Wiley & Sons, Ltd.