To gain insight into the mechanisms which govern cellular identity in the intestinal epithelium we have begun a detailed study of the murine cdx-2 homeobox gene. We isolated and sequenced both cDNA and genomic clones in order to define the open reading frame and mature transcript. A detailed analysis of cdx-2 transcript levels late in embryogenesis showed that they increased 6-7-fold at a time when the gut undergoes a major developmental transition. In the adult, cdx-2 was expressed in colon in a region-specific manner with transcripts some 5-fold more abundant in the cecum as compared with the rectum. In situ hybridization and immunohistochemical experiments showed cdx-2 transcripts and protein were present in all epithelial cells in the proximal colon irrespective of their degree of differentiation. In distal colon, however, transcripts were most abundant in undifferentiated cells at the bottom of crypts, whereas the highest protein levels were present in mature cells in the upper half of crypts. We conclude that cdx-2 is expressed specifically in gut epithelium where it is not restricted to a particular cell lineage. Rather, the rostrocaudal expression gradient suggests that it may play a role in specifying positional identity.