Under the influence of estradiol, the vaginal epithelium undergoes a process of growth and differentiation known as cornification. To analyze the biosynthetic changes that underlie this phenomenon in rats, it was necessary to first isolate the vaginal epithelium from hormonally stimulated animals. This was accomplished by two different methods: enzymatic digestion with a trypsin/pancreatin solution, which produced a disaggregated population of cells from the living epithelial layers; or disruption of basement membrane adhesion with a chaotropic salt solution (2 M NaSCN), which produced intact, full thickness tissue sheets. Extended extraction of the cells and sheets with an alkaline urea buffer followed by analysis of these extracts on denaturing polyacrylamide gels revealed that both preparations contained six major polypeptides in the mol wt range of 50,000-60,000 daltons. Differential solubilization indicated that these proteins are keratin subunits, and amino acid analysis confirmed this for the 53,000-dalton polypeptide (53K) purified from preparative sodium dodecyl sulfate gels. On two-dimensional gel electrophoresis, 53K produced a group of three acidic spots (alpha, beta, and gamma), with isoelectric points of 4.6, 5.5, and 5.4, respectively. Partial peptide maps revealed that these spots are isoelectric variants of the same protein. Immunization of rabbits with 53K produced a keratin antiserum that was primarily directed against the immunogen and cross-reacted weakly with a 50,000- and a 57,000-dalton band. This work demonstrated the feasibility of obtaining well defined molecular probes from a hormone-responsive epithelial system.