Azospirillum brasilense is a nitrogen-fixing, root-colonizing bacterium that brings about plant-growth-promoting effects mainly because of its ability to produce phytohormones. Ethylenediamine (EDA)-resistant mutants of A. brasilense were isolated and screened for their higher ability to decrease acetylene and release ammonia in the medium. One of the mutants showed considerably higher levels of acetylene decrease and ammonia excretion. Nitrogenase activity of this mutant was relatively resistant to inhibition by NH4Cl. Adenosine triphosphate ribosylation of dinitrogenase reductase in the mutant did not increase even in presence of 10 mM NH4Cl. Although the mutant showed decreased glutamine synthetase (GS) activity, neither the levels of GS synthesized by the mutant nor the NH4+-binding site in the GS differed from those of the parent. The main reason for the release of ammonia by the mutant seems to be the fixation of higher levels of nitrogen than its GS can assimilate, as well as higher levels of adenylylation of GS, which may decrease ammonia assimilation.