Seven strains of the genus Kluyveromyces were screened for beta-galactosidase activity and Kluyveromyces marxianus ATCC 16045 was selected as the best enzyme producer for culture medium optimization. The production of beta-galactosidase by submerged cultivation was evaluated using a factorial design and response surface methodology. The culture medium containing whey and parboiled rice effluent was formulated to maximize the production of beta-galactosidase. The effects of the initial pH and the concentrations of whey lactose, peptone, (NH4)(2)SO4, yeast extract, and parboiled rice effluent on enzyme production were studied using a 2 (IV) (6-2) fractional design. A CCRD (24 trials plus axial and central points) was used for the four variables selected from the fractional design (lactose, peptone, (NH4)(2)SO4 and yeast extract), with beta-galactosidase activity as the response. The optimum conditions established for production were a whey (lactose) concentration of 120 g/L, a yeast extract concentration of 5 g/L, a peptone concentration of 15 g/L, a (NH4)(2)SO4 concentration of 15 g/L, a parboiled rice effluent concentration of 30 g/L, and a pH value of 4.0. Under these conditions, the highest enzymatic activity of 10.4 U/mL was measured, being 9.5-9.7 as the values predicted by the proposed model, showing an enzymatic activity increase of 30% using alternative sources of lactose and nitrogen for beta-galactosidase production.