A synthetic pyruvate:H(2) pathway was constructed in Escherichia coli BL21(DE3) by co-expression of six proteins: E. coli YdbK, Clostridium pasteurianum [4Fe-4S]-ferredoxin, and Clostridium acetobutylicum HydF, HydE, HydG, and HydA. The effect of cofactor addition and host strain on H(2) yield and fermentation product accumulation was studied, together with in vitro reconstitution of the entire pathway. The deletion of iscR and/or the addition of thiamine pyrophosphate to the medium enhanced the total and specific activity of recombinant YdbK and increased the yield of H(2) per glucose. It was concluded that the introduced pathway outcompeted other pyruvate-consuming reactions, and that the ability to compete for pyruvate at least in part was determined by total YdbK activity. The results demonstrate the successful construction of a high-yielding H(2) pathway in a microorganism that effectively does not synthesize any H(2). The additional co-expression of Bacillus subtilis AmyE enabled starch-dependent H(2) synthesis in minimal media.