Thymic stromal cell lines, termed MRL104 and MRL28, have been isolated from long-term liquid cultures of thymic stromal cells from MRL/l mice. The capacities of these parental lines and derived clones to express Ia antigens and to produce cytokines involved in T-cell proliferation and/or differentiation were investigated. Parental lines and their clones did not exhibit a typical fibroblastic, macrophage-like, or epithelial appearance in electron as well as phase-contrast micrographs. These thymic stromal cells seemed to differ from established fibroblast lines in that these thymic stromal cells expressed Ia antigens after exposure to interferon-gamma (IFN-gamma), whereas fibroblast lines did not. They also appeared to differ from macrophage cell lines in that they lacked the expression of Mac-1 antigens on their cell surface and produced no detectable level of interleukin-1 (IL1) before or even after exposure to lipopolysaccharide. When these parental lines and its clones were tested for their ability to produce various types of cytokines, it was revealed that they were capable of producing colony-stimulating factor (CSF), IL6, and thymic stroma-derived T cell growth factor (TSTGF), which was recently described, but were unable to generate other lymphokines and IFNs. Thus these cell lines and clones represent unique features in that they have potentials to express Ia antigens and to produce CSF, IL6, and TSTGF. The biological significance for the expression of these features is discussed in the context of intrathymic T-cell maturation and T-cell repertoire selection.