Azospirillum brasilense was treated with nitrosoguanidine and five machete resistant mutant strains were isolated. The effect of machete on growth, N₂-fixation, cell constituents, hydrogenase activity, nitrogenase activity, glutamine synthetase, and nitrate reductase activities were studied. Mutant strains MHT 3 and MHT 5 showed lesser nitrate uptake and exhibited greater growth, hydrogenase activity (H₂ uptake), G.S. activity, and N₂ase activity in nitrogen free medium than parental and other mutant strains at 30°C compared with 42°C. Inoculation of rice with A. brasilense and mutant strains led to a significant increase in nitrogenase activity and grain yield compared with the uninoculated control with and without machete (handweeded). Differential response of strains was noted on the nitrogen contents of plants and seeds with significant strains × genotype interaction. These mutants were stable and retained their resistance even after several subcultures through medium free of machete.