To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new 15N-labeled N2, detection methodology, which is free from interference from atmospheric N2 contamination. 30N2 (15N15N) and 29N2 (15N14N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N2 gas having natural abundance of 15N (0.366 atom%) as a carrier gas. The detection limit was 0.04% 30N2, and the linearity extended at least to 40% 30N2. When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with 15NO3-, denitrification product (30N2) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype.