The growth and immunologic characteristics of in vitro cultured thymic epithelial cells from NZB, NZW, NZB/W, BALB/c, DBA/2, and N:NIH(S) mice were studied. Cultures were prepared from irradiated (400 R) mice to prevent in vitro autosensitization of thymocytes by epithelial elements, thereby permitting selective growth without the attendant hazards of local cytotoxicity; epithelial cells were enriched and fibroblasts were removed by treatment with a trypsin-EDTA mixture. Forty-eight hours after initial plating, BALB/c, DBA/2, and N:NIH(S) mice all demonstrated a proliferation of large PAS-positive and PAS-negative epithelial cells in a mosaic like arrangement and within 30 days of culture greater than a 95% enrichment of such epithelial cells was achieved. In contrast, proliferation of epithelial cells from thymic explants of New Zealand mice disclosed a marked age dependent relationship. In particular, explants of NZB thymus, age 1 to 52 weeks, failed to produce growth of any cellular elements except fibroblasts. There was, however, evidence of epithelial cell proliferation of NZB/W thymic explants derived from mice less than 8 weeks of age. Nonetheless, serial observations of clones of epithelial cells from NZB/W mice demonstrated vacuolization and degeneration within 2 to 8 weeks of culture, suggesting a pre-programmed defect. Similarly, whereas isolated thymic epithelial cells from BALB/c or young NZB/W mice induced an elevation of thy 1.2 on spleen cells and an increase in responsiveness of nude spleen cells to the T cell mitogen, Con A, cultures from NZB or older NZB/W mice were ineffective. Furthermore, incubation of 7-month-old NZB spleen cells, but not lymph node cells, with a BALB/c thymic epithelial cell monolayer significantly restored the ability to respond to Con A. Moreover, the abnormal proliferation, measured by DNA synthetic response, of NZB spleen seeking but not lymph node seeking thymocytes injected into lethally irradiated C57BL/6 mice was suppressed by preincubation with thymic epithelial cells. Finally, thymic epithelial cells enhanced the ability of NZB thymocytes to suppress a graft versus host reaction. We suggest that functional alterations of thymic epithelial cells in New Zealand mice may be critical to the primary development of autoimmunity and that several of the lymphoid cell defects in these mice, including suppressor cell function, can be corrected by thymic epithelial cells.