The non-T accessory cell requirement for cytolytic T lymphocyte (CTL) generation in vitro was studied in a system which makes use of particulate membrane preparations as a source of alloantigen, and spleen cells from alloimmune mice as a source of responding cells. It is shown that removal of nylon-adherent cells from the responding cell population strongly reduced CTL generation, whereas direct removal of Ig+, phagocytic or plastic-adherent cells had no effect. The CTL response of the nylon-nonadherent cell population could be reconstituted by the addition of normal spleen cells, which by themselves do not generate CTL in response to particulate alloantigen. The accessory cell function of normal spleen cells was not affected by depletion of T cells or of phagocytic cells, but was sensitive to gamma-irradiation (1000 rd). The system thus demonstrates the requirement for a nylon-adherent accessory cell population in the secondary CTL response to particulate alloantigens which does not exhibit the typical characteristics of T cells, B cells or macrophages.