Effect of components in the medium on the induction of the nitrogen-fixing activity in the free-living state of slow-growing soybean nodule bacteria, B. japonicum, was studied using five strains each of Hup+ and Hup-. Nitrogenase activity was expressed regardless of the oxygen concentration in a defined solid medium (GM) while glutamine, malic acid and molybdenum were found to be the essential components for the expression of the nitrogenase activity. Uptake hydrogenase activity was also expressed on the same nitrogenase induction medium (GM). Effect of the medium composition on the expression of the uptake hydrogenase activity was different from that for the nitrogenase activity. Glutamine, malic acid and molybdenum, were not essential for the expression of the uptake hydrogenase activity. This newly developed method on defined solid medium can be used for the evaluation of the nitrogen-fixing activity of strains in Bradyrhizobium.