Annexin A2 is a member of the annexin family of Ca(2+)-dependent lipid binding proteins and believed to be engaged in membrane transport processes in a number of cell types. In small intestinal enterocytes, we localized annexin A2 to the brush border region, where it was found mainly on the lumenal side of the microvilli, showing an apical secretion by a "nonclassical" mechanism. In addition, annexin A2 was associated with surface-connected, deep apical tubules in the apical terminal web region and with an underlying pleiomorphic, tubulo-vesicular compartment (subapical compartment/multivesicular bodies). By subcellular fractionation, the 36 kDa full-length form of annexin A2 was approximately equally distributed between the Mg(2+)-precipitated fraction (containing intracellular and basolateral membranes) and the microvillar membrane fraction. In addition, a 33 kDa molecular form of annexin A2 was seen in the latter fraction that could be generated from the full-length annexin A2 by digestion with trypsin. Taken together, the results suggest that annexin A2 acts in exocytic apical membrane trafficking and is proteolytically cleaved in situ by pancreatic proteinases once it has become externalized to the lumenal side of the brush border membrane. On the basis of its well-known membrane fusogenic properties, we propose a model for the nonclassical membrane translocation of annexin A2.