Metabolic flows, especially those of amino acids, were determined and analyzed at different stages of a batch fermentation for microbial transglutaminase production by Streptoverticillium mobaraense. The method is mainly based on mass balances and measurements of amino acids and other metabolites. The measurements included the consumption rate of glucose, intake rates of all amino acids and production rates of carbon dioxide, cell mass, and transglutaminase. Three groups of metabolic flows were determined by three different methods. Those in the first group are determined by solely using measurement results. The second group deals with the synthesis of most amino acids. The metabolic flows were determined by using a mass-balancing method considering the contribution of these amino acids to the synthesis of cells and product, i.e., transglutaminase. The third group includes the reactions covering all other important intermediates. The metabolic flows in this group were calculated by a metabolite-balancing method Metabolic flows during different fermentation phases were thus determined. The distribution of metabolic flows of amino acids implies that growth and transglutaminase production are active as long as there are free amino acids available in the medium. An important factor which limits further growth and production is probably the cross-linking action of transglutaminase on the nitrogen source in the medium. The results suggest that a nitrogen source other than peptone and/or amino acids might improve growth and production.