In order to develop a fermentation process for lactase (β-D-galactosidase) production, we selected an excellent lactase producer, Kluyveromyces lactis. KY 5466, from our yeast culture collection. Some of its mutant derivatives which formed a blue pigment from 5-bromo-4-chloro-3-indolyl-β-D-galactoside in the presence of glucose and those which assimilated phenyl-β-Dgalactoside as a carbon source produced 2 to 2.7 times as much lactase as the parent strain. In the late stage of cultivation, the lactase activity decreased to zero for all strains tested soon after the complete consumption of sugar. This phenomenon was found to be correlated with a decrease in the efficiency of protein extraction from the cells. The maximal amount of lactase produced reached 155 units per ml at 48 hr in a 5-1 jar fermentor culture with sugar feeding.