Cloned H-2b-restricted male antigen-specific cytotoxic T cells were kept in culture in the presence of IL 2 for over 2 yr. During this time, they lost their antigenic specificity and now exhibit high NK-like activity. Three separate clones HY 1, HY 2, and HY 3 and their subclones, when tested on a panel of NK-sensitive and NK-insensitive target cells, showed typical lysis patterns of NK cells. Three cytotoxicity-loss mutants were obtained by subcloning. At least one of these cytotoxicity-loss mutants still exhibited binding to NK-sensitive target cells. The various HY clones were compared with cloned alloreactive cytotoxic T cells of B10 anti-B10.D2 specificity with respect to surface markers, target specificity, kinetics of lysis, morphology, and influence of monoclonal antibodies and chemicals on cytotoxicity. Only one major difference between cloned NK-like and alloreactive T cells was detected: monoclonal anti-Lyt-2 antibodies inhibited cytotoxic activity of alloreactive T cells but not of NK-like cells, although both expressed Lyt-2 on their surface. Morphologically, HY cells were characterized by numerous lysosomal granules in their cytoplasm. Clones with NK-like activity differed from cytotoxicity-loss mutants by both ultrastructural appearance and enzyme histochemistry of lysosomes. Interaction of NK-like cells with YAC-1 cells induced membrane lesions of variable size in target cells. Effects of chemicals influencing lysosomal pathways such as monensin, NH4Cl, and chloroquine on cytotoxicity were investigated. Monensin as well as NH4Cl inhibited cytotoxicity of NK as well as alloreactive clones, whereas chloroquine did not. Spleen cells from acutely virus-infected mice with high NK reactivity showed the same behavior except for chloroquine, which was slightly inhibitory. The addition of monensin to the cells resulted in morphologic changes in the lysosomal granules. These data strongly suggest that NK-like killing is triggered by unknown substance(s) released from lysosomes of effector cells. Cloning of antigen-specific cytotoxic T cells under conditions of limiting dilution and continuous growing in medium containing Con A supernatant but not the relevant antigen often leads to loss of specificity and appearance of NK-like activity. The results presented are representative of a great amount of work in this and probably many other laboratories attempting to clone cytotoxic effector T cells.