The distribution of the major keratin mRNAs expressed during terminal differentiation and fetal development of the rat intestinal epithelium has been examined by in situ hybridization. We have obtained and characterized a partial cDNA clone encoding human keratin 20 whose sequence spans from the coil la region through the 3' poly(A) tail. Sequence data and immunoblot analysis demonstrated that keratin 20 is the human homologue of the rat keratin 21, suggesting the existence of a single type I keratin specifically expressed by differentiated intestinal epithelial cells. Four cRNA probes, specific for keratins 8, 18, 19, and 20 respectively, were prepared and found to specifically hybridize with their respective mRNA species from total intestinal RNA preparations. Analysis of frozen tissue sections by in situ hybridization revealed that, in the adult intestine, keratin 18 and 19 mRNAs are restricted to the region of the crypts, keratin 8 mRNA is found along the entire crypt-villus axis, and keratin 20 mRNA is expressed only by the differentiated villus cells. This pattern is established late during fetal rat intestinal development: in the undifferentiated stratified epithelium present at 16 days gestation (16dg) mRNAs coding for keratins 8, 18, and 19 are expressed by all epithelial cells and keratin 20 mRNA is absent. Upon completion of villus formation at 20dg (2 days before birth) keratin 18 and 19 mRNAs become strictly confined to cells at the base of the nascent villi and we observed the appearance of keratin 20 mRNA which, like keratin 8 mRNA, is expressed by the entire epithelium. These results strongly suggest that transcriptional regulation of keratin genes in the intestinal epithelium occurs at the level of both immature and terminally differentiated epithelial cells, and is tightly regulated during both fetal development and crypt-to-villus differentiation of the intestinal epithelium.