A method is described for the three-dimensional (3-D) in vitro culture of nontransformed gastrointestinal epithelial cells from the human duodenal mucosa. Biopsies obtained from human duodenum were finely minced. The tissue fragments were suspended in culture medium supplemented with 5% fetal calf serum and the appropriate antibiotics. The suspended mucosal fragments generated spheroid-like multicellular vesicles consisting of highly prismatic absorptive and goblet cells retaining most of the histological features of the tissue in vivo. We performed immunocytochemical studies to determine the origin of the vesicles using monoclonal antibodies against EP4. The histochemistry of the vesicles showed alkaline phosphatase activity. Ultrastructural studies revealed that these cells exhibit characteristics of normal duodenal cells in vivo: apical microvilli, glycocalyx, tight junctions and desmosomes, lateral membrane interdigitations, mucous droplets, and a well-developed Golgi apparatus. An overgrowth of the vesicles by fibroblasts was not seen during cultivation. In contrast with the two-dimensional cell cultures grown on artificial supports, the vesicle cells show organization similar to that of natural epithelia. The polarization and cytoarchitecture of normal gastrointestinal epithelial cells cultured as 3-D vesicles are comparable to those known for the native tissue. This study was undertaken to provide a morphological baseline for subsequent infection experiments.