The research field for applications of lactose hydrolysis has been investigated for several decades. Lactose intolerance, improvement for technical processing of solutions containing lactose, and utilization of lactose in whey are the main topics for development of biotechnological processes. We report here the optimization of a hollow-fiber membrane reactor process for enzymatic lactose hydrolysis. Lactase was circulated abluminally during luminal flow of skim milk. The main problem, the growth of microorganisms in the enzyme solution, was minimized by sterile filtration, ultraviolet irradiation, and temperature adjustment. Based on previous experiments at 23 +/- 2 degrees C, further characterization was carried out at 8 +/- 2 degrees C, 15 +/- 2 degrees C (beta-galactosidase), and 58 +/- 2 degrees C (thermostable beta-glycosidase) varying enzyme activity and flow rates. For a cost-effective process, the parameters 15 +/- 2 degrees C, 240 U/mL of beta-galactosidase, an enzyme solution flow rate of 25 L/h, and a skim milk flow rate of about 9 L/h should be used in order to achieve an aimed productivity of 360 g/(L x h) and to run at conditions for the highest process long-term stability.