Hyperpolarized NMR is a promising technique for non-invasive imaging of tissue metabolism in vivo. However, the pathways that can be studied are limited by the fast T1 decay of the nuclear spin order. In metabolites containing pairs of coupled nuclear spins-1/2, the spin order may be maintained by exploiting the non-magnetic singlet (spin-0) state of the pair. This may allow preservation of the hyperpolarization in vivo during transport to tissues of interest, such as tumors, or to detect slower metabolic reactions. We show here that in human blood and in a mouse in vivo at millitesla fields the (13) C singlet lifetime of [1,2-(13) C2 ]pyruvate was significantly longer than the (13) C T1 , although it was shorter than the T1 at field strengths of several tesla. We also examine the singlet-derived NMR spectrum observed for hyperpolarized [1,2-(13) C2 ]lactate, originating from the metabolism of [1,2-(13) C2 ]pyruvate. © 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.