In this study, we analyze the regulation of the squamous-specific gene, cornifin α, by estrogen and retinoic acid in vaginal and uterine epithelial cells. In ovariectomized animals, the vaginal epithelium consists of a stratified, nonkeratinizing epithelium which changes into a highly-stratified, keratinizing epithelium upon treatment with estradiol. This transition is accompanied by a dramatic induction of the crosslinked envelope precursor, cornifin α. An increase in cornifin mRNA can be detected as early as 3 h after treatment. A similar effect is observed for the synthetic estrogenic agent diethylstilbestrol while other steroid hormones, including testosterone, progesterone or dexamethasone have little effect on cornifin expression. In contrast to the vagina, estradiol induces neither squamous differentiation nor expression of cornifin α in the uterine epithelium. Similar to the action of estradiol, vitamin A-deficiency greatly enhances squamous differentiation and keratinization in the vaginal epithelium. But unlike estradiol, it induces squamous metaplasia in the normally columnar, uterine epithelium, which eventually is replaced by a keratinizing epithelium in severe deficiency. This transition is associated with an induction of cornifin a expression. Immunohistochemical and in situ hybridization analysis localizes cornifin protein and mRNA in the suprabasal layers of the squamous epithelium. Our results demonstrate that estrogen and retinoids play key roles in the regulation of differentiation and cornifin α expression in the uterine and vaginal epithelium.