The improvement of the yield of hydrogen at fermentative production using molasses as a substrate was investigated from two points of view; i.e. firstly, through the control of production pathway and secondly, by the condition of nutrient source for growing. It was shown that succinate, one of the products of Enterobacter aerogenes strain E.82005, was produced inductively by accumulation of CO2 which was produced simultaneously with acetate, ethanol and other products. The amount of residual NADH which is the base material of H-2 evolution increased when Ar or H-2 was blown into the culture liquid. From these results, it was concluded that the removal of CO2 from culture liquid effected the promotion of the yield of hydrogen. It was also shown that the existence of sufficient nitrogen source for bacterial growth was a necessary condition to get entire yield of hydrogen. The combination of experiments to remove CO2 and to give sufficient nitrogen source was found to yield 1.58 mol hydrogen from 1 mol sugar in terms of glucose.