ABSTRACTHydrogen metabolism was studied in the anaerobic bacterium, Sporomusa sp. strain DMG 58, by measuring natural abundance levels of deuterium in H2, H2O, and individual fatty acids during acetogenic growth on H2/CO2. Four cultures were grown, each in medium with a distinct hydrogen‐isotopic composition (δD‐H2O). The δD value of H2 was quantified in the residual gas exiting the growth chambers and found to decrease concurrently with net H2 consumption, indicating rapid isotope exchange between H2 and H2O. An isotopic mass balance was used to constrain the efficiency with which H2 was activated by the cell and the reducing equivalents catabolized, which we term the H2 utilization efficiency. Results indicate that H2 utilization efficiency in these cultures is less than 20% during the growth phase, and less than 2% after the growth phase. The gross rate of cellular H2 activation was similar in the growth phase and afterward. Biomass harvested at the end of each experiment was used to analyse the D/H of individual membrane lipids. Values of δD were highly correlated between lipids and water (δD‐lipids = 0.59 × δD‐water – 381‰; R2 = 0.995), indicating the source of lipid hydrogen is in isotopic equilibrium with water. Results are consistent with two possibilities: (i) water is the sole source of hydrogen to lipids, and the fractionation during biosynthesis is significantly larger than previously observed (α = 0.59), or (ii) hydrogen from H2 is incorporated into lipids, but only after reaching isotopic equilibrium with H2O. Fatty acids were strongly depleted in deuterium relative to all other organisms studied thus far, and such large depletions may prove useful as biomarkers for studying H2 cycling in anoxic environments as well as in the geological record.