Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL(-) R. sphaeroides produced 2.42 l H-2/l culture and 0.25 l H-2/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H-2/l culture and 0.21 l H-2/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL- R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photo-heterotrophic H-2 production by R. sphaeroides. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.