The present study describes a mechanism that regulates the activation of cytotoxic T lymphocytes (CTL) derived from natural killer (NK) cells. In the absence of antigenic stimulation, polyclonal activation of cytotoxic lymphocytes was induced by culturing the splenic responders with exogenous peritoneal macrophages and indomethacin. The effectors were characterized as T cells, whereas the precursors were of NK origin. To activate these NK-derived CTL, although there was no need for direct contact between responding precursors and macrophages, the role of macrophages could not be substituted by the addition of exogenous IL 2, macrophage supernatant, or IL 1. In contrast, supernatant from syngeneic lymphocyte-macrophage cultures with indomethacin induced CTL generation. This finding indicates that other lymphokines might also be produced and that they are essential for CTL activation. The need for indomethacin, a prostaglandin (PG) synthetase inhibitor, indicated that PG also played a regulatory role. The addition of 1 X 10(-9) M to 1 X 10(-8) M exogenous PGE2 to the cultures in the first 24 to 48 hr completely suppressed CTL activation. Our results clearly show that through regulation by macrophages and PG, NK-derived precursors differentiated into mature CTL. A different receptor repertoire appeared to be present in different clones of NK precursors. In the absence of antigenic stimulation, removal of the restriction by PG allowed the macrophages or their products to interact with the lymphocyte to produce various lymphokines. These lymphokines further signaled the NK precursors to be polyclonally activated and to differentiate into CTL.