The sensitivity of human intestinal lactase to pancreatic proteases was tested both in vitro and in vivo. Lactase specific activity in brush border membranes was decreased by 26%–27% during incubation with trypsin at pH 7.0 in patients with normal intestinal lactase levels, whereas in patients with lactase deficiency the inactivation was 75%. However, when lactase levels from deficient patients' mucosa were increased relative to trypsin during incubation so that they were comparable to the levels of activity in normal mucosa, inactivation of lactase in deficient patients was only 45%. Therefore, in these patients the greater in vitro lactase inactivation by trypsin could be explained at least in part by an increased trypsin/lactase ratio. Sucrase levels were decreased in vitro by trypsin (about 40%), but maltase activity was unaffected. The effect of pancreatic proteases was tested in vivo in patients with pancreatic insufficiency. After the addition of pancreatic enzymes (Viokase), lactase specific activity fell by 16% in patients with normal lactase, and by 38.5% in patients with lactase deficiency. In both groups of patients, lactase levels fell to a greater extent than did sucrase or maltase. These data demonstrate that pancreatic proteases can alter intestinal lactase activity in humans. Moreover, in lactase-deficient patients, lactase activity decreases to a greater extent than in patients with normal lactase, resulting in further deficiency of this enzyme.