The isolation and characterization of a Rhizobium etli glutamate auxotroph, TAD12, harbouring a single Tn5 insertion, is reported. This mutant produced no detectable glutamate synthase (GOGAT) activity. The cloning and physical characterization of a 7.2 kb fragment of R. etli DNA harbouring the structural genes gltB and gltD encoding the two GOGAT subunits GltB and GltD is also reported. In comparison with the wild-type strain (CFN42), the GOGAT mutant strain utilized less succinate and glutamate and grew less with this and other amino acids as nitrogen source. R. etli assimilates ammonium by the glutamine synthetase (GS)-GOGAT pathway and a GOGAT mutant prevents the cycling of glutamine by this pathway, something that impairs nitrogen and carbon metabolism and explains the decrease in the amino-nitrogen during exponential growth, with glutamate as nitrogen source. GOGAT activity also has a role in ammonium turnover and in the synthesis of amino acids and proteins, processes that are necessary to sustain cell viability in non-growing conditions. The assimilation of ammonium is important during symbiosis and glutamate constitutes 20-40% of the total amino-nitrogen. In symbiosis, the blockage of ammonium assimilation by a GOGAT mutation significantly decreases the amino-nitrogen pool of the bacteroids and may explain why more N(2) is fixed in ammonium, excreted to the plant cell, transported to the leaves and stored in the seeds.