The thymuses of 14-day-old CBA mouse embryos cultured for 14 days in organ cultures supplemented with mouse serum contained lymphocytes that were highly reactive to the mitogens concanavalin A (Con A) and leukoagglutinin (LA) and to irradiated allogeneic spleen cells in vitro. In contrast, thymocytes derived from fetal-calf-serum-supplemented organ cultures displayed much weaker responses to the two mitogens and little or no response to the allogeneic spleen cells. Control thymocytes obtained from 1-month-old CBA mice responded well to Con A but not to LA. The responses of these cells to allogeneic spleen cells were delayed compared with the organ-culture-derived cells, which responded promptly, with a maximum already on culture day 2-3. The results demonstrate that the organ culture system provides a convenient model for further study of the generation of immunoreactive cells in the mouse thymus.