The stable hydrogen isotope composition of persistent biomolecules is used as a paleoenvironmental proxy. While much previous work has focused on plant leaf wax-derived n-alkanes, the potential of bacterial and archaeal lipid biomarkers as carriers of H isotope signatures remains underexplored. Here we investigated H isotope distributions in the membrane lipids of the ammonia-oxidizing chemoautotroph Nitrosopumilus maritimus strain SCM1. Hydrogen isotope ratios were measured on the biphytane chains of tetraether membrane lipids extracted from steady-state continuous cultures cultivated at slow, medium, and fast growth rates. In contrast to recent work on bacterial fatty acids, where the direction and magnitude of isotopic fractionation varies widely (ca. 600 ‰ range) in response to the choice of substrate and pathways of energy metabolism, archaeal biphytane data in the present work are relatively invariant. The weighted average 2H/1H fractionation values relative to growth water (2εL/W) only ranged from 272 to 260 ‰, despite a three-fold difference in doubling times (30.8 hr to 92.5 hr), yielding an average growth-rate effect of 0.2 ‰ hr-1. These 2εL/W values are more depleted than all heterotrophic archaeal and bacterial lipid H isotope measurements in the literature, and on par with those from other autotrophic archaea, as well as isoproenoid-based lipids in photoautotrophic algae. N. maritimus values of 2εL/W also varied systematically with the number of internal rings (cyclopentyl + cyclohexyl), increasing for each additional ring by 6.4 ± 2.7 ‰. Using an isotope flux-balance model in tandem with a comprehensive analysis of the sources of H in archaeal lipid biosynthesis, we use this observation to estimate the kinetic isotope effects (KIEs) of H incorporation from water; from reducing cofactors such as ferredoxin, and for the transhydrogenation reaction(s) that convert the electron-donor derived NADH into NADPH for anabolic reactions. Consistent with prior studies on bacteria, our results indicate the KIEs of reducing cofactors and transhydrogenation processes in archaea are highly fractionating, while those involving exchange of water protons are less so. When combined with the observation of minimal growth-rate sensitivity, our results suggest biphytanes of autotrophic 3HP/4HB Thaumarchaeota may be offset from source waters by a nearly constant 2εL/W value. Together with the ring effect, this implies that all biphytanes originating from a common source should have a predictable ordering of their isotope ratios with respect to biphytane ring number, allowing precise reconstruction of the original δ2H value of the growth water. Collectively, these patterns indicate archaeal biphytanes have potential as paleo-hydrological proxies, either as a complement or an alternative to leaf wax n-alkanes.