The intracellular routes taken by aminopeptidase N, an apically expressed enzyme in the enterocyte, was followed in small intestinal cultures of pig using either immunoelectron microscopy (immunogold labeling) or continuous labeling with [35S]methionine. Aminopeptidase N was found in the microvillar membrane, the Golgi complex, apical small smooth vesicles, and various acidic lysosomal/endosomal-like organelles. By culturing mucosal explants in the presence of either cycloheximide or (3-(2,4-dinitroanilino)-3-amino-N-methylpropylamine) (DAMP) it was demonstrated that the apical small smooth vesicles are exocytotic and that the low pH in the acid compartments is of no importance for intracellular transport and correct sorting of aminopeptidase N. Furthermore, our results show that the majority of the aminopeptidase N in the lysosomal/endosomal-like compartments is newly synthesized.