In our search for a biologic role for lymphokine-activated killers (LAK), we examined their generation from murine thymocytes. Normal adult thymocytes were capable of generating LAK upon culture with relatively large doses (500 to 1000 U/ml) of interleukin 2. Normal thymocytes were fractionated into four subsets by virtue of their co-expression of the Lyt-2 and L3T4 markers: Lyt-2+ L3T4+ (2+4+); 2+4-; 2-4+; and 2-4-. None of these subsets had any natural killer activity. Upon examining the ability of these subsets to generate LAK, it was found that the 2-4- subset was the most potent and required the smallest relative amounts of interleukin 2. In addition to lysing tumor cells, thymus-derived LAK were capable of killing "fresh" 2+4+ thymocytes. Fresh 2+4-, 2-4+, 2-4-, and cortisone-resistant thymocytes were resistant to lysis by LAK. Upon mitogen stimulation, however, the cortisone-resistant thymocytes and 2+4- thymocytes became LAK-susceptible. These data demonstrate a possible mechanism for the elimination of the 2+4+ thymocyte subset which is generally believed to be a "dead-end" population. Moreover, these data suggest a possible biologic role for LAK in the process of thymocyte maturation and intrathymic selection.