To examine the multiple stages of lipoprotein packaging during development, we studied localization, ontogeny, and regulation of microsomal transfer protein (MTP), a crucial protein for lipid transport. With the use of immunofluorescence, MTP was identified in villus and crypt epithelial cells in different regions of human fetal intestine, including colon. Staining was detected as early as the 13th wk of gestation in all gut segments and was almost entirely confined to the columnar epithelial cells of the jejunum and colon. Unlike immunofluorescence, which provides qualitative but not quantitative information on MTP signal, enzymatic assays revealed a decreasing gradient from proximal small intestine to distal, as confirmed by immunoblot. Activity of MTP in small intestinal explants cultured for different incubation periods (0, 4, 8, and 24 h) peaked at 4 h but remained insensitive to different concentrations of oleic acid. Also, a trend toward increasing MTP activity was observed at 20-22 wk of gestation. Finally, in strong contrast to jejunal efficiency, colonic explants displayed impaired lipid production, apolipoprotein biogenesis, and lipoprotein assembly, in association with poor expression of MTP. These findings provide the first evidence that human fetal gut is able to express MTP and emphasize the distinct regional distribution, regulation by oleic acid, and ontogeny of MTP.