Disaccharides 2-O-, 3-O-, and 4-O-beta-D-galactopyranosyl-D-xyloses (2, 3, and 1, respectively) were obtained by beta-galactosidase-catalyzed reactions for their use in the evaluation of intestinal lactase activity in vivo. Their administration to suckling rats followed by determination of the derived D-xylose in the urine and measurement of lactase activity in intestinal homogenates showed 1 to be the most suitable disaccharide for a potential test of the deficiency of intestinal lactase. The synthesis of 1 was further studied by evaluating the effect of different variables on the yield and regioselectivity of the enzymatic galactosylation, and the purification process was optimized.