AbstractA fast and sensitive procedure to determine the turnover of the H2 hydrogen of lactate and quantify its 2H‐enrichment by 1H NMR is illustrated using C6 cells metabolizing (3‐13C) lactate in 50% 2H2O (vol/vol). 2H substitution of the lactate H2 hydrogen resulted in two easily detectable transformations of the vicinal H3 doublet resonance: 1) the formation of an H3 singlet due to the disappearance of the homonuclear coupling to H2 (3JβH‐αH = 7.0 Hz), and 2) an upfield isotopic shift derived from the vicinal 2H2 substitution (Δ3 = –0.007 ppm). Only those lactate molecules that have passed through the cell cytosol experience these effects, since H2 deuteration involves lactate dehydrogenase activity and NAD(2H). Thus, analysis of the observed shifted and unshifted H3 lactate resonances from the incubation medium allows the discrimination of the perprotonated (3‐13C) lactate added as substrate, and the (3‐13C, 2‐2H) lactate recycled to the incubation medium after passage through the cytosol. Magn Reson Med, 2005. © 2005 Wiley‐Liss, Inc.