The effect of deleting the genes encoding the twin-arginine translocation (Tat) system on H2 production by Escherichia coli strain MC4100 and its formate hydrogenlyase upregulated mutant (ΔhycA) was investigated. H2 evolution tests using two mutant strains defective in Tat transport (ΔtatC and ΔtatA-E) showed that the rate doubled from 0.88±0.28mL H2mg dry weight−1L culture−1 in the parental strain, to 1.70±0.15 and 1.75±0.18mL H2mg dry weight−1L culture−1, respectively, in the ΔtatC and ΔtatA-E strains. This increase was comparable to that of a previously characterized hydrogen over-producing E. coli strain carrying a ΔhycA allele. Construction of a tatC, ΔhycA double deletion strain did not increase hydrogen production further. Inactivation of the Tat system prevents correct assembly of the uptake hydrogenases and formate dehydrogenases in the cytoplasmic membrane and it is postulated that the subsequent loss of basal levels of respiratory-linked hydrogen and formate oxidation accounts for the observed increases in formate-dependent hydrogen evolution.