Rhodobacter sphaeroides KCTC 12085 is a natural isolate which shows a higher ability of photoheterotrophic H-2 production compared with a laboratory strain R. sphaeroides 2.4.1. The H-2 production by R. sphaeroides is observed only under light conditions. In order to render R. sphaeroides to produce H-2 fermentatively as well, a 4.4-kb Rhodospirillum rubrum DNA encompassing a gene coding for pyruvate-formate lyase (PFL) with its putative maturation protein, and a 29.4-kb R. rubrum gene cluster including formate-hydrogen lyase (FHL) complex, its maturation proteins, a transcriptional activator for FHL, and Fe-only hydrogenase with its maturation proteins were cloned and mobilized into R. sphaeroides KCTC 12085. The recombinant R. sphaeroides evolved H-2 during dark fermentative growth. The H-2 production by the recombinant R. sphaeroides under photoheterotrophic conditions was elevated twofold to show 4 mol H-2/mol of glucose compared with 2 mol H-2/mol of glucose by its parental strain carrying empty vector. Interestingly, addition of hypophosphite, an inhibitor of PFL, to the recombinant R. sphaeroides under photoheterotrophic conditions still resulted in 3 mol H-2/mol of glucose, suggestive of active H-2 production not only by nitrogenase but also by Fe-only hydrogenase, which are insensitive to hypophosphite. The results further suggest the photoheterotrophic H-2 production by FHL unless hypophosphite is present. Thus, the H-2 production by the recombinant R. sphaeroides under photoheterotrophic conditions appears to be mediated by the concerted actions of FHL, Fe-only hydrogenase, and nitrogenase. The H-2-evolving activity by the recombinant R. sphaeroides containing Fe-only hydrogenase of R. rubrum appears to depend on the presence its maturation proteins, suggestive of lack of such proteins in R. sphaeroides. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.