This study is on the investigation of hydrogen production and substrate removal by photosynthetic bacteria. After using of Rhodospillum rubrum KS-301 and IFO 3986, which are photosynthetic bacteria as strains, R. rubrum KS-301 was turned out a better strain. And result of experiment in which glucose and sodium lactate, components of wastewater, were used limiting substrates, showed that the productivity of hydrogen was indifferent with the kind of substrates. In batch experiments using free cells and immobilized whole cells, the decrease in hydrogen productivity was observed in the latter case. From the results of these experiments, specific growth rate of cells, specific utilization rate of glucose, and specific production rate of hydrogen were calculated. And each rate was expressed in the form of Monod equation of which parameters were estimated. Also the optimum condition of hydrogen production for free cells was $30^{circ}C$, pH 7, and 12,000 Lux, and the optimum immobilized condition was as follows: initial immobilized cell concentration 1.0g/L, sodium alginate concentration 2% and light intensity 12,000 Lux.