Some amino acids (alanine, asparagine, glutamate, glycine, proline, and tyrosine) were used as nitrogen sources in combination with carbon sources (succinate and malate) to study growth properties and H 2 production by purple non-sulfur bacterium Rhodobacter sphaeroides strains A-10 and D-3. Both strains produced H 2 in succinate–glutamate and malate–glutamate media. Succinate was a better carbon source than malate. In comparison with strain D-3, strain A-10 was able to utilize proline, alanine or tyrosine as nitrogen sources in succinate medium and to produce H 2 . Both strains were unable to produce H 2 in the presence of asparagine or glycine as nitrogen sources. N,N′-dicyclohexylcarbodiimide, the F 0 F 1 -ATPase inhibitor, led to marked inhibition of H 2 production activity of R. sphaeroides. The results suggest that the R. sphaeroides cells growth can be achieved by the use of a large diversity of substrates but only some of them can increase the H 2 production rate.