AbstractThe subsurface process of serpentinization creates an extreme environment for microbial life. This environment includes reducing, ultra‐basic groundwater that is limited in electron acceptors. Despite these challenging conditions, there is a great deal of potential energy available to support microbial metabolisms both in the anaerobic subsurface and in aerobic surface environments where serpentinization associated groundwater discharges. In this study, the available energy was quantified through the calculation of chemical affinities, Ar, for three sites of active serpentinization in North America: the Tablelands (NL, CAN), The Cedars (CA, USA), and Aqua de Ney (CA, USA). The results showed that Ar values for each reaction were similar for all sites studied; however, the available energy varied a great deal from reaction to reaction. For example, the reaction of carbon monoxide oxidation provided the most energy to the system followed closely by hydrogen oxidation and methanotrophy. Potential microbial metabolisms were tested, simulating surface and subsurface conditions, in a laboratory‐based setting using microcosms with materials from each site. During these microcosm experiments, carbon monoxide oxidation was not observed, and there was little evidence of methane oxidation. Unexpectedly, microbial methanogenesis was observed in methane oxidation microcosms using material collected from The Cedars. The microbial production of methane occurred despite the addition of electron acceptors demonstrating the broad tolerance of methanogens at The Cedars for less reducing conditions.