Nonspecific cytotoxicity developed reproducibly and with high frequency in limit dilution cultures consisting of low numbers of murine cells stimulated with concanavalin A in the presence of growth factors and irradiated filler cells. The individual clones in cultures showing nonspecific killing were all derived from single, Thy-1+, Ly-2+ cells. At early times of culture (day 5 or 6), clones appeared to be specific in their lytic activity, as expected of cytolytic T lymphocytes (CTL). On continued culture (day 8 or 9), most of the originally specific CTL clones became nonspecific, killing a range of murine target cells, both syngeneic and allogeneic. The lack of specificity was observed at all effector cell doses. The effector cells responsible for the nonspecific cytolysis were Thy-1+ and Ly-2+, as were most cells in the cultures. The effector cells had the normal DNA content for a dividing T cell population, and most cells in the cultures had a normal chromosome complement. In mixed cultures in which the responder cells and the irradiated filler cells were from different mouse strains, the nonspecific killers displayed the Thy-1 and H-2 allotypes of the responder, and not of the filler cells. The development of a broad cytotoxic potential appears to be a normal and rapid event when Ly-2+ T cell-derived CTL-clones are grown under these conditions; this is a caveat for the use of limit dilution cultures to determine the T cell specificity repertoire. The relationship between these nonspecific CTL, activated lymphocyte killers, and natural killer cells is discussed.