Keratin 14 (K14) is believed to play a pivotal role in the maintenance of epidermal cell shape and contributing to their resistance to mechanical trauma, thereby protecting the cells from lysing. Mice harboring a K14 null mutation produce phenotypic characteristics of epidermolysis bullosa simplex, a skin blistering disease (Lloyd et al., 1995, J Cell Biol 129: 1329–1344). K14 null animals die several days after birth, making the detailed study of the consequences of K14 deletion in epidermal cell physiology in vivo particularly difficult. To define the consequences of K14 loss more precisely, we used an in vitro approach by isolating K14−/− cell lines and studying epidermal differentiation in the K14 null background. Several keratinocyte cell lines were generated from 6-day-old mice homozygous for a targeted disruption of the K14 gene (lines designated MKC-5, MKC-23, and MKC-33) and from their wild-type littermates (lines designated MKC-1 and MKC-6). Under low Ca 2+(0.066 mM) and low serum (2%) conditions, both wild-type and mutant cells were able to adhere to collagen type I–coated dishes and form epithelial sheets. They maintained basal epidermal cell characteristics and continued to proliferate without obvious signs of terminal differentiation; however, K14−/− cells proliferated two-to threefold slower than did their wild-type counterparts. The distribution of K5, the natural partner of K14, at the immunofluorescence level was also normal looking in the K14−/− MKC-5 cells, but with fewer filaments detectable, consistent with the approximately 20% reduction in K5 detectable on immunoblots. K17 expression was increased approximately 40% in the K14−/− cells. The levels of K15 and K16 were not different in the MKC-5 and MKC-6 cell lines, suggesting that they are not contributing factors to the stabilization of K5 in the mutant cells. K8, K19, and vimentin were undetectable in both lines. Both MKC-5 and MKC-6 cells underwent morphological and biochemical differentiation in response to a switch to high Ca 2+ medium. These findings indicate that K14−/− MKC-5 cells preserve the morphological, biochemical, and physiological characteristics of epidermal cells for an extensive period of time in vitro, likely due to the compensatory expression of K17. The culturing capacity of these cells also permits the analysis of keratinocyte growth and differentiation in the absence of K14. In addition, the culturing methods we describe will be useful for the generation of epithelial cell lines from a wealth of increasingly available knockout mouse strains with early lethality. J. Cell. Physiol. 180: 409–421, 1999.© 1999 Wiley-Liss, Inc.