The thymic stromal microenvironment is required for the generation of immunocompetent T lymphocytes. However, the different thymic stromal cell types have not been fully characterized and their roles regarding T-cell development are not completely understood. To address the phenotypic characteristics of the epithelial component of the human thymic microenvironment as well as its functional involvement in T-cell development, we have established cloned thymic epithelial cell (TEC) lines from fetal and postnatal human thymuses by an explant technique, repeated subculture, and limiting dilution cloning. These cloned TEC lines were shown to be derived from cortical epithelium and to express a number of cell-surface molecules including CD40, major histocompatibility complex (MHC) HLA-ABC and HLA-DR antigens, homing- associated cell-adhesion molecule (H-CAM), intercellular adhesion molecule-1 (ICAM-1), leukocyte function-associated antigen 3 (LFA-3), and beta 1 subfamily integrins. Finally, both postnatal and fetal TEC clones were shown to produce interleukin-1 alpha (IL-1 alpha), IL-6, and IL-7. These well-defined cloned TEC lines may provide useful tools for the study of TEC biology and for the understanding of the precise role played by TEC in human T-cell development.