Using water as the substrate and driven by solar energy, cyanobacteria have immense potential for renewable H₂ production. However, the H₂-production reaction catalyzed by the Synechocystis bi-directional [NiFe]-hydrogenase (Hox) does not sustain in the presence of O₂, the latter is an inherent byproduct of oxygenic photosynthesis. One approach to surmount this challenge is to transfer an O₂-tolerant hydrogenase into this cyanobacterium for sustained H₂ production. Prior research in our laboratory has uncovered from the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS a [NiFe]-hydrogenase with a half life near 21 hours when whole cells were stirred in ambient air. Via transposon mutagenesis, we have cloned six hydrogenase genes in Rx. gelatinosus, including those encoding the large (CooH) and small (CooL) subunits. Moreover, we also identified six accessory genes (hypABCDEF) with predicted functions in the [NiFe]-active site assembly. We have cloned nearly all of these coo and hyp genes downstream of inducible promoters in E. coli BL21. Western blots indicated that the hydrogenase large and small subunits were indeed synthesized. However, a lack of hydrogenase activity above that of background suggested that the [NiFe] active site was not assembled properly in the E. coli host. We have since created a hydrogenase-deficient E. coli host strain that possesses T7 polymerase, thus providing a clean background for controlled expression of foreign hydrogenases. Similarly, we have also created a hydrogenase-knockout strain of Synechocystis to eliminate background activity. Work is ongoing to optimize hydrogenase expression both in E. coli and in the Synechocystis host.