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In vitro experiments of small intestinal mucosal function and metabolism utilizing excised tissue have been limited to a few hours by rapid epithelial cell necrosis which occurs with current incubation methods. We describe a method for culturing human mucosal biopsies for up to 24 hr employing organ culture methodology and demonstrate its potential application to studies of mucosal function. Peroral biopsies were placed in organ culture plates and maintained with modified Trowell's medium in 95% O2-5% CO2 at 37°C for 6-24 hr. To study cell proliferation, 2 μc of thymidine-3H was added per ml of medium. To study fat absorption, biopsies were exposed to micellar solutions of linolenic acid, monoolein, and taurodeoxycholate in Krebs-Ringer buffer for 15 min after culture in vitro for 24 hr. After 24 hr of culture, villi were shorter and wider. Cells in the lamina were reduced in number. Light and electron microscopic morphology of epithelial cells compared favorably to those of control biopsies except in occasional areas of partial necrosis. Some absorptive cells were more cuboidal and contained more lysosomes; many appeared entirely normal. Most crypt cells appeared normal; some contained increased glycogen and lysosomes. Mitoses were present, and labeled cells were abundant in crypts of biopsies after 6 hr of incubation with thymidine-3H-containing medium. By 24 hr. labeled cells migrated to the base of the villi. When biopsies cultured in vitro were subsequently exposed to micellar lipid, numerous lipid droplets were identified in the cytoplasm of absorptive cells. Thus, after 24 hr in vitro under these culture conditions, many human small intestinal epithelial cells maintain near normal morphology, epithelial cell proliferation proceeds, and fat absorption occurs.