Stimulation of cells from long-term primary MLC with Con A resulted in the generation of CTL activity comparable in magnitude to that induced by reexposure of the cells to the original stimulating cellular antigen. CTL generated by stimulation of long-term MLC cells with ConA had lytic activity specific for the original stimulating alloantigen used in primary MLC. The pattern of stimulation of long-term MLC cells with Con A differed from that of restimulation with alloantigen in that there was no detectable CTL activity the first 24 hr after Con A stimulation and the peak lytic activity occurred later. Unlike restimulation with alloantigen early lytic activity after Con A stimulation was dependent on DNA synthesis. PHA also proved to be an effective agent for stimulating cytolytic activity in long-term MLC cells. The response to PHA was comparable in magnitude to that generated by Con A. Stimulation of long-term MLC cells with T cell mitogens gave decreased cell recoveries relative to restimulation with alloantigen, however, the lytic activity per cells recovered was generally greater in the mitogen-stimulated cultures.