The effects of different carbon and nitrogen sources on the growth, nitrogenase activity, and carbon metabolism of Gluconacetobacter diazotrophicus were investigated. The amino acids asparagine, aspartic acid, and glutamic acid affected microbial growth and nitrogenase activity. Several enzymatic activities involved in the tricarboxylic acid cycle were affected by the carbon source used. In addition, glucose and gluconate significantly increased the oxygen consumption (respiration rate) of whole cells of G. diazotrophicus grown under aerobic conditions. Enzymes responsible for direct oxidation of glucose and gluconate were especially active in cells grown with sucrose and gluconate. The presence of amino acids in the apoplastic and symplastic sap of sugarcane stems suggests that these compounds might be of importance in the regulation of growth and nitrogenase activity during the symbiotic association. The information obtained from the plant-bacterium association together with the results of other biochemical studies could contribute to the development of biotechnological applications of G. diazotrophicus.