In the later stages of a batch fermentation for microbial transglutaminase production by Streptoverticillium mobaraense the availability of a nitrogen source accessible to the microorganism becomes critical. Fed-batch fermentation is investigated with the aim of avoiding this substrate limitation. When peptone is used as a nitrogen source in the feed, no significant improvement of growth and transglutaminase production is observed. This is probably due to crosslinking of the nitrogen source by the transglutaminase produced. Using an inorganic nitrogen source alone does not give satisfactory growth and production. A fed-batch fermentation method has thus been developed to deal with this problem. In the batch phase of the fermentation, an initial medium containing peptone, designed on the basis of the stoichiometric requirements of the microorganism, is used to ensure optimal growth. In the feeding phase, ammonium sulphate is used instead to avoid the crosslinking effect. The feed composition, mainly the amount of nitrogen and carbon source, is also based on the stoichiometric requirements of the organism, taking into account the replacement of peptone by ammonium sulphate. By using this fed-batch fermentation technique. cell-mass dry weight and transglutaminase production could be increased by 33% and 80% respectively, compared to those in a batch fermentation.