The murine homeobox gene Cdx-1 is postulated to play an important role in the process of cellular commitment in the intestinal epithelium based upon its graded, tissue-specific expression pattern in the adult animal. We have isolated and sequenced both cDNA and genomic clones in order to understand how this expression pattern is regulated and to determine the amino acid sequence of the encoded protein. Our studies show that the Cdx-1 gene contains 3 exons and 2 introns and encodes a 268-amino acid protein which is translated from a 1.7-1.8-kilobase mRNA. Transcription of the Cdx-1 gene initiates some 30 base pairs downstream from a typical TATA box which, together with the 5' end of the gene, is located within a CpG island. Transient transfection experiments have shown three regions which regulate Cdx-1 gene expression. These include a typical silencer element at -589 to -380, an element at -887 to -1040 which blocks the effect of the silencer and a positive element at -47 to +66; the only one which shows epithelial cell specificity. Using gel shift and footprinting assays we have detected proteins in nuclear extracts prepared from colonic epithelial cells which bind to the silencer element. These initial results suggest that Cdx-1 gene expression is regulated by multiple positive and negative transcription factors.