The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G(0)) and N-Z Case TT (NZ(0)) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 L-f/mL), achieved at G(0) = 9.7 g/L and NZ(0) = 43.5 g/L, was 80% higher than that obtained with standard cultivations (G(0) = 8.0 g/L and NZ(0) = 25.0 g/L).