The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5‘-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5‘-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.