In Rhodobacter capsulatus, the hupL gene encoding the large subunit of the uptake-hydrogenase (Hup) enzyme complex was mutated by insertion of an interposon. The mutant neither synthesized an active hydrogenase nor grew photoautotrophically. Under conditions of nitrogen (N) limitation, photoheterotrophic cultures of the wild type and the mutant evolved H-2 by activity of the nitrogenase enzyme complex. When grown with glutamate as an N source and either D,L-malate or L-lactate as carbon sources, the efficiency of H-2 production by the HupL mutant was higher than 90%, whereas wild-type cultures exhibited efficiencies of 54% (with D,L-malate) and 64% (with L-lactate), respectively. With NH4+ as the N source, efficiencies of H-2 production were 70% (mutant) and 52% (wild type).