Abstract Mouse thymic epithelial cell lines (MTEC) were established from Day 14-18 fetal thymus by two novel protocols. The first protocol involved the selection of TEC by the formation of complexes with adult thymocytes after transformation with the helper-free Ad5.SVR4 recombinant virus. The second protocol involved the stimulation and selection of TEC in Ca 2+-free medium by the formation of complexes with Day 14 fetal thymocytes. The resulting TECs formed several types of thymic epithelial clusters to which Day 14 fetal thymocytes could bind. Many of these fetal thymocytes could deeply infiltrate, colonize, and proliferate within the clusters of NCAM high LFA-1 low TEC (MTSC-0420-1.4, MTSC-0420-1.5, and MTSC-0613-1.2 clusters), whereas very few could infiltrate the clusters of NCAM low LFA-1 high TEC (MTSC-0531-5.1 and MTSC-0531-5.2) and none bound to or infiltrated an NCAM neg fibroblast cluster (MTSC-fibro.). Hence, it is possible that the NCAM-positive epithelial cell lines are derived from the thymus cortex, where they may play an important role in intrathymic migration and the clonal growth of pro-T cells in fetal thymus.