Epithelial cells derived from a variety of glandular and other nonkeratinized rat tissues (pituitary, thyroid, bladder, endometrium, trachea, seminal vesicle, prostate, and mammary epithelium) were serially cultivated using a feeder layer of lethally irradiated 3T3 cells. The epithelial cells grew as progressively expanding colonies, in some cases stratified, and were shown to form cornified envelopes upon ionophore-induced activation of cross-linking. Cultures derived from each tissue were distinguishable from the others by characteristic cellular appearance and colony morphology. Those examined in greater detail could be distinguished biochemically in three ways. (a) A majority of cells in sparse cultures of bladder, tracheal, endometrial, and vaginal epithelial cells were capable of envelope formation, whereas those from pituitary, thyroid, seminal vesicle, and mammary epithelia did not attain maximal envelope forming ability until after confluence. (b) Bladder, thyroid, and pituitary cells exhibited different electrophoretic profiles of keratins, which accounted for 20-50% of the cellular protein. (c) Bladder cells were distinguished from thyroid and pituitary cells by a greater suppression of envelope-forming ability by vitamin A. These observations showed that cells from many epithelia have the potential to express properties of keratinocytes in culture while maintaining morphological and physiological differences. Serial passage of these cells generated continuous lines.