The diazotrophic bacterium, Herbaspirillum seropedicae can be used as inoculant in grasses as an alternative to nitrogen fertilizers. Studies of its general metabolism and specially its nitrogen metabolism, will contribute to a better exploitation of its potential as inoculant. The ntrYX genes, first described in Azorhizobium caulinodans, play an important role in the nitrogen metabolism of this organism once mutation on these genes leads to a reduction of 50 to 80% in nitrogen fixation and deficiency of growth in nitrate as the sole nitrogen source. The ntrYX genes had been identified during the H. seropedicae genome sequencing (GENOPAR Program). The H. seropedicae ntrY mutant strain A01 exhibited a nitrogenase activity similar to the wild strain and also the GS activity was not altered by the mutation in this gene. The A01 strain (ntrY^-) is not able to grow on nitrate as the sole nitrogen source, but grew well with ammonium, glutamate, aspartate, asparagine, glutamine, serine, alanine and urea as nitrogen source. The promoter region upstream to the ntrY gene is functional and showed the highest expression level when grown on nitrate. The RNA polymerase alternative s⁵⁴ factor seems to be necessary for expression of these genes that also requires the NtrC protein for activation. Mutation on the ntrY gene of H. seropedicae affected the expression of genes involved with assimilatory (nasFEDCBA) and respiratory metabolism of nitrate and nitrite (narKnirBDC and narXL). These results indicate that ntrY gene of H.seropedicae is involved with the expression of the genes related with both assimilatory and respiratory nitrate metabolism.