Keratinocytes were cultured from adult and newborn rat epidermis using the 3T3 feeder cell technique. By modifying culture conditions a long-lived line of newborn rat keratinocytes was developed which showed a plating efficiency of 40% and a doubling time of 16 h. The cells produced stratified colonies with tonofilaments, desmosomes, cell envelopes, and keratohyaline granules. When the cells were grown on a collagen gel they formed a thick stratum corneum and many keratohyaline granules. The fibrous proteins synthesized by the newborn rat cultured keratinocytes were different than those of newborn rat epidermis but similar to those of adult rat cultured keratinocytes. A histidine-rich basic protein was identified by immunologic techniques but it appeared to be more heterogeneous than that of newborn rat epidermis. A cell envelope precursor protein was identified by dansyl cadaverine incorporation studies and was identical to a major envelope precursor of newborn rat epidermis. The growth characteristics, colony morphology, and biochemical markers did not change for up to 40 passages and there was no evidence of malignant transformation. Because of their case of growth and long-term survival these cells are useful for studying a variety of problems related to keratinization.