Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. The reversible (bi-directional) hydrogenase (hoxEFUYH) of Synechocystis sp. PCC 6803 is a [NiFe]-type enzyme that produces hydrogen via the reaction 2H+ + 2e- ↔ H2 (g); the source of the two electrons is NADH. Hydrogenase enzymes in E. coli are involved in two distinct modes of hydrogen metabolism: hydrogen production via hydrogenase 3 (encoded by hycABCDEFGHI) and hydrogen uptake by hydrogenase 1 (encoded by hyaABCDEF) and hydrogenase 2 (encoded by hybOABCDEFG). A study was conducted to determine how the cyanobacterial locus increases hydrogen production. Different components of the cyanobacterial locus were expressed. Overexpression of HoxU alone (small diaphorase subunit) accounted for 43% of the additional hydrogen produced by HoxEFUYH. Hydrogen production in TG1/pBS(Kan)HoxUYH was somewhat better than that in TG1/pBS(Kan)Synhox indicating the importance of proteins HoxU, HoxY, and HoxH. This is an abstract of a paper presented at the AIChE Annual Meeting (Salt Lake City, UT 11/4-9/2007).