NAD(P)H:H2 pathways are theoretically predicted to reach equilibrium at very low partial headspace H2 pressure. An evaluation of the directionality of such near-equilibrium pathways in vivo, using a defined experimental system, is therefore important in order to determine its potential for application. Many anaerobic microorganisms have evolved NAD(P)H:H2 pathways; however, they are either not genetically tractable, and/or contain multiple H2 synthesis/consumption pathways linked with other more thermodynamically favourable substrates, such as pyruvate. We therefore constructed a synthetic ferredoxin-dependent NAD(P)H:H2 pathway model system in Escherichia coli BL21(DE3) and experimentally evaluated the thermodynamic limitations of nucleotide pyridine-dependent H2 synthesis under closed batch conditions. NADPH-dependent H2 accumulation was observed with a maximum partial H2 pressure equivalent to a biochemically effective intracellular NADPH/NADP+ ratio of 13:1. The molar yield of the NADPH:H2 pathway was restricted by thermodynamic limitations as it was strongly dependent on the headspace : liquid ratio of the culture vessels. When the substrate specificity was extended to NADH, only the reverse pathway directionality, H2 consumption, was observed above a partial H2 pressure of 40 Pa. Substitution of NADH with NADPH or other intermediates, as the main electron acceptor/donor of glucose catabolism and precursor of H2, is more likely to be applicable for H2 production.