We examined optimum culture conditions of Rhodopseudomonas sphaeroides B5 for effective utilization of substrate and sunlight for hydrogen production. The optimum concentration range of DL-lactate as electron donor for hydrogen production by resting cells was from 5 to 50mM, and optimun CN ratio (lactate/glutamat) for maintenence of hydrogen production activity by growing cultures was from 5 to 6. Hydrogen production by the cultures of low cell density (0.36mg/ml dry cells) was saturated with 10 Klux light intensity. Under constant illumination of 50Klux which was set up as the average medium value of annual variation of sunlight intensity, hydrogen production with various cell densities in the culture resulted in highest production rate (132${mu}$l/hr/mg dry cells) up to 0.64mg/ml dry cells. However, the amount of total hydrogen production was saturated with cell density of 2.1mg/ml dry cells. In addition to these, the optimum inner thickness pervious to light of the culture vessel for hydrogen production which was measured under sunlight was 5 cm.