A simple culture technique is described for obtaining homogeneous populations of differentiating epidermal cells from adult amphibian skin. This population of cells continues its normal differentiation, namely keratinization, in vitro. By the third day of culture more than 99-9% of the cells in the population contain immunoreactive keratin. During the next 5-7 days of culture these cells synthesize increasing amounts of a tissue-specific keratin-like protein until this protein constitutes more than 25% of the total extractable carboxymethylated proteins of these cells. Under the conditions described cell division does not occur during the course of differentiation. When vitamin A is added to the culture medium the rate of keratinization is decreased; when its antagonist citral is added, keratinization is accelerated. These factors influence only the rate but not the direction of differentiation.