A random transposon-based mutagenesis system was optimized for the purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS. Screening for hydrogenase-deficient phenotypes resulted in the isolation of six independent mutants in a mini-Tn5 library. One of the mutations was in a gene showing high amino acid sequence similarity to HypF proteins in other organisms. Inactivation of hydrogen uptake activity in the hypF-deficient mutant resulted in a dramatic increase in the hydrogen evolution capacity of T. roseopersicina under nitrogen-fixing conditions. This mutant is therefore a promising candidate for use in practical biohydrogen-producing systems. The reconstructed hypF gene was able to complement the hypF-deficient mutant of T. roseopersicina BBS. Heterologous complementation experiments, using hypF mutant strains of T. roseopersicina, Escherichia coli, and Ralstonia eutropha and various hypF genes, were performed. They were successful in all of the cases tested, although for E. coli, the regulatory region of the foreign gene had to be replaced in order to achieve partial complementation. RT-PCR data suggested that HypF has no effect on the transcriptional regulation of the structural genes of hydrogenases in this organism.