Abstract The in vivo release of a microvillar enzyme, aminopeptidase N (EC 184.108.40.206), and a cytosolic enzyme, lactate dehydrogenase (EC 220.127.116.11), into the intestinal lumen was measured to gain information on the fraction of desquamated cell protein in intestinal juice and on the mechanism of release of intestinal microvillar enzymes. 1.6% and 2.9% of the mucosal activities of aminopeptidase N and lactate dehydrogenase were released to the intestinal lumen per hour, respectively. The ratios between aminopeptidase N and lactate dehydrogenase in intestinal perfusates and mucosal homogenates were similar. This result is compatible with the view that aminopeptidase N in the rat small intestine is predominantly released into the intestinal lumen by desquamation of enterocytes. This conclusion was supported by the failure to demonstrate microvasculation of the microvilli by electron microscopy. 30–40% of the aminopeptidase N in the intestinal lumen is membrane-bound indicating that partial solubilization occurs during desquamation. The addition of calcium ions did not augment the release of aminopeptidase N or the membrane-bound fraction in the lumen. 10–20% of the protein content in the intestinal lumen is due to extruded cell protein. This includes aminopeptidase N which constitutes 1% of the luminal protein.