Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ; The effects of sucrose concentration, pH, temperature and aeration rate on the production of biomass and inulinase (2,1-beta-D-fructan fructanohydrolase, EC 3.2.1.7) were investigated using fed-batch fermentation by Kluyveromyces marxianus var. bulgaricus. The best sucrose concentration for enzyme activity was 10 g L-1, whereas activity was inhibited when the sucrose concentration was 20 g L-1. Enzyme activity was strongly influenced by pH of the culture. pH 5.0 was the most suitable for both enzyme activity and cell growth, achieving 98.04 U mL-1 and 14.05 g L-1, respectively, after 72 hours of cultivation. The highest production of inulinase and biomass was observed at temperatures of 25 degrees C and 30 degrees C, whereas a decrease in activity was observed at 35 degrees C and 40 degrees C. The best aeration rate was 1.0 vvm. The increase in aeration rate or oxygen injection provided no increase in enzyme activity and biomass.