Photosynthetic microbes can produce the clean-burning fuel hydrogen using one of nature's most plentiful resources, sunlight. Anoxygenic photosynthetic bacteria generate hydrogen and ammonia during a process known as biological nitrogen fixation. This reaction is catalyzed by the enzyme nitrogenase and consumes nitrogen gas, ATP and electrons. One bacterium, Rhodopseudomonas palustris, has a remarkable ability to obtain electrons from green plant-derived material and to efficiently absorb both high and low intensity light energy to form ATP. Manipulating R. palustris, or a similar organism, to produce hydrogen commercially will require us to identify all its genes that contribute to hydrogen production and to understand how this process is regulated in cells. We have obtained mutant strains of this bacterium for which growth depends on hydrogen production. In these mutants metabolism is redirected such that cells use nitrogenase as an electron sink and hydrogen-producing enzyme, and not as a catalyst for ammonia synthesis. We have used the mutants to show that, in addition to nitrogenase genes,18 genes outside of the nitrogenase gene cluster may contribute to hydrogen production. Our results demonstrate that photosynthetic bacteria can be genetically manipulated for sustained production of pure hydrogen in a variety of cultivation conditions in the absence of oxygen, nitrogen or other gases as long as light and an electron donor are supplied.