Mouse thymic stromal cells growing in vitro have been stained with fluorescent antibodies to identify the cells found in these cultures. By means of anti-cytokeratin antibody 35 beta H11, it could be shown that nearly all the cells in the cultures were epithelial. Three other antibodies which bind to specific regions of the thymus were used to detect subpopulations of the epithelial cells. Cells growing as confluent carpets stained with antibody ER-TR5, which is specific for medullary epithelium. Antigens for two other antibodies, MD2 (known to label cells at the medullary side of the cortical-medullary junction) and CDR1 (which reacts with cortical epithelium) were expressed on the cultured cells only after the addition of exogenous signals. MD2 antigen was expressed on a very small percentage of the cells incorporated into networks as well as a few dispersed cells, and seemed related to the activity of fibroblasts. CDR1 antigen appeared on the majority of the cells organized into networks and those dispersed in the cultures. Gamma-interferon (IFN-gamma) and IL-2, lymphokines usually associated with T-cell reactivity, were found to be involved in its expression. It was possible to isolate the CDR1 subpopulation from the cultures by means of the antigen present on the surface of these cells.