Molecular hydrogen (H (sub 2) ) is a central metabolite that couples organic matter degradation and terminal electron-accepting processes. H (sub 2) levels in natural environments are often regulated by microbial syntrophy; therefore, pore-water H (sub 2) concentration is a useful parameter for studying biogeochemical processes in sediments. However, little is known about H (sub 2) concentrations in marine subsurface sediments. Previous studies applying either a headspace equilibration technique or an extraction method for the analysis of pore-water H (sub 2) in deeply buried sediments have generated results that sometimes contradict the principles established based on studies of microbial culture and surface sediments. In this study, we first evaluated and optimized an extraction method, which was then applied in combination with a headspace equilibration method to determine concentrations of pore-water H (sub 2) in subseafloor sediments along a transect of five sites of different water depths and geochemical regimes at the continental margin off Namibia, SE Atlantic. The two methods generated depth profiles with some similarities in curve shape, but the extraction method yielded higher H (sub 2) values than the headspace equilibration technique. By comparing the two data sets with thermodynamic calculations of potential terminal electron-accepting processes, we were able to provide a first evaluation of syntrophic conditions in subseafloor sediment from the perspective of H (sub 2) biogeochemistry. We observed that in the sulfate reduction zone, the H (sub 2) concentrations are higher than the H (sub 2) threshold allowed for the next most favorable terminal metabolism (methanogenesis), suggesting relaxation of coupling between H (sub 2) -producing and H (sub 2) -consuming activities at these depths. In contrast, the H (sub 2) concentrations in the upper methanogenic zone are low enough for methanogens to outcompete CO (sub 2) -reducing acetogens. Our findings suggest the existence of varied extents of syntrophic H (sub 2) coupling in subseafloor sediment. Abstract Copyright (2012) Elsevier, B.V.