Cultures of thymic epithelial cells were generated and maintained in valine-free minimum essential medium (MEM) supplemented with 690 mg/liter of D-valine. These cultures have been maintained for 1 year through multiple passages by trypsinization of 60-70% confluent monolayers. Large and small epithelial cells were present in early cultures. They were separated into two stable subpopulations based on (1) their differential growth rates and (2) their differential adherence to the culture substratum. These morphologically distinct cell populations, TECS and TECL, were 100% keratin positive and contained cells with desmosomes and tonofilaments, all characteristics of epithelial cells. Esterase analysis of both cell populations revealed a 1 and 9% esterase-positive cell population in cultures of keratin-positive small (TECS) and large (TECL) cells, respectively. The percentages of esterase-positive cells corresponded to the 2 and 10% populations of TECS and TECL, respectively, that contained both desmosomes and phagolysosomes. These results establish conditions for the long-term propagation of pure thymic epithelial cells. Such cultures can be used to study the functional interactions between epithelial cells and lymphoid cells. Morphologic and histochemical analyses have identified subsets of these cells which may prove to have differential effects on thymocyte proliferative and developmental processes.