This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed-batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ(0)) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50-60 L-f/mL) for NZ(0) > 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield (similar to 60 L-f/mL) about 33% higher than those of fed-batch runs with addition at fixed concentration (similar to 45 L-f/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial,scale. The results of this work promise to substantially improve the present production of tetanus toxin and may be adopted for human vaccine production after detoxification and purification. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 88-92, 2010