Lactase (β- d -galactosidase) was produced by Candida pseudotropicalis grown in deproteinized whey. Maximum enzyme production in 2% whey was obtained by supplementation with 0.15% yeast extract, 0.1% (NH 4 ) 2 SO 4 , and 0.05% KH 2 PO 4 (wt/vol). Highest enzyme values (4.35 U/mg of cells and 68 U/ml) were obtained with 10 to 12% whey, while enzyme yield was maximal in 2% whey (0.87 U/mg of whey). Optimal initial pH for cultivation was 3.5. The best conditions for extraction included 2% (wt/vol) chloroform, 10 h of treatment, pH 6.6 and higher, and 30 to 37°C. Optimum pH and temperature for enzyme activity were 6.2 and 47°C. The enzyme had a K m for O -nitrophenyl-β- d -galactopyranoside of 3.06 × 10 −3 M and the initial V max was estimated as 6.63 × 10 −8 M per min. It hydrolized 50 and 100% of the lactose in whey and milk within 4 and 5 h, respectively, at 37°C. The lyophilized enzyme retained 95% of activity for 3 months when stored at −20°C.