A lactose-free, low-cost culture medium for the production of beta-D-galactosidase by Kluyveromyces marxianus was formulated. At high aeration rates (2.2 vvm) and concentrations of 100 g sugar cane molasses l(-1) as carbon source and 100 g corn steep liquor l(-1) as vitamin and nitrogen source an enzyme production of 708 U l(-1) h was achieved. This was 20% higher than using a medium that contained lactose which is considered the primary inductor of beta-D-galactosidase synthesis.