Thymuses of 14-day-old mouse embryos could be maintained in organ culture, using serum-free medium, with generation of lymphocytes that showed strong proliferative reactivity to allogeneic spleen cells in mixed lymphocyte cultures (MLC). Such organ culture derived thymocytes also developed cytotoxic activity against target cells carrying the alloantigens of the stimulating spleen cells in the MLC. The generated cytotoxic activity was, however, proportionately lower than that seen with thymocytes of young mice. A time course study showed that cells with apparent reactivity to both allogeneic spleen cells and to the mitogen Concanavalin A (Con A) first appeared in the thymus organ cultures on day 8-9.