Seven monoclonal antibodies were prepared against cytoskeletal components of rat intestinal brush borders. In the following paper (Chandler, J. S., Calnek, D., and Quaroni, A., J. Biol. Chem. 266, 11932-11938), three of them were shown to be specific for, respectively, keratin 8 (RK4), keratin 19 (RK7), and a newly identified type I keratin (keratin 21) (RK5). With these antibodies we have investigated the changes in keratin gene expression accompanying intestinal cell differentiation. Keratin 21 was detected exclusively in differentiated villus cells and in goblet, enteroendocrine, and Paneth cells in the crypts; in the proliferative crypt cells keratin 19 was predominant. Analysis of keratins expressed by cultured rat crypt cells (IEC cells) confirmed the absence of keratin 21 in undifferentiated intestinal cells. Changes in keratin's expression similar to those observed with cell differentiation in the adult intestinal mucosa were also demonstrated during early fetal intestinal development: the stratified epithelium present at 15-16 days of gestation contained predominantly keratin 19 with only a small amount of keratin 8; keratin 21 was first detected at 18-19 days of gestation, concomitant with the appearance of a well formed brush border and an apical cytoplasmic terminal web. These results suggest that keratin tonofilaments may play a role in the morphological and structural alterations accompanying intestinal cell differentiation in vivo.