The production of beta-galactosidase by Kluyveromyces fragilis was studied in different culture systems, with dissolved oxygen concentration control and using defined media. An operating strategy of fed-batch culture with automatic control of substrate addition regulated by dissolved oxygen concentration, consisting of the replacement of variable volumes of broth by fresh medium (once the fed-batch culture has finished), was designed. The volumetric enzyme productivity (Q(p), 13 600 UI dm(-3) h(-1)) obtained was 38% higher than that reached in continuous culture of K fragilis with dissolved oxygen concentration control and far higher than that obtained by batch culture of K fragilis under the same aeration conditions.