Various metal ions play a key role in biohydrogen (H2) production by phototrophic bacteria through incorporation into or stimulating the responsible enzymes and/or related pathways. The Ni (II) and Mg (II) ions effects on growth and H2 production by Rhodobacter sphaeroides strain MDC6521 isolated from mineral springs in Armenia were established. The highest growth specific rate was obtained with 4-6聽渭M Ni2+ and 5聽mM Mg2+. pH of the growth medium changed from 7.0 to 9.2-9.4 during the bacterial growth up to 72聽h in spite of Ni2+ added but pH increased in different manner with Mg2+. In the presence of 2-4聽渭M Ni2+ external oxidation-reduction potential (ORP) decreased to more negative values (鈭?00聽卤聽15聽mV). This decrease of ORP indicated 鈭?.7-fold enhanced H2 yield (9.80聽mmol聽L鈭?) with Ni2+ compared with the control (without Ni2+). The H2 yield determined in the medium with Mg2+ was 鈭?.2 fold higher than that with 1聽mM Mg2+. These results reveal new regulatory ways to improve H2 production by R. sphaeroides those were depending on Ni2+ and Mg2+ of different concentrations.