T lymphocytes expressing the surface phenotype Lyt-2- L3T4- represent a minor population of immature thymocytes that appear to be the precursors of mature T cells. Cells with the same apparent surface phenotype also accumulate in vast numbers in the lymphoid tissues of the autoimmune lpr mouse. Lyt-2- L3T4- T lymphocytes from lpr lymph node (LN) or normal thymus express low to undetectable levels, respectively, of surface antigen receptor. In addition, they produce reduced amounts of lymphokines compared with normal T cells and lack precursors of alloantigen-specific cytolytic T lymphocytes. We previously showed that after culture with phorbol esters and interleukin 2, lpr Lyt-2- L3T4- T lymphocytes proliferate and differentiate, acquiring increased levels of surface antigen receptor by most cells, as well as Lyt-2 by a portion. We now show that cultured Lyt-2- L3T4- T cells from lpr LN or normal thymus are very efficiently cytolytic toward not only allogeneic tumor targets, but also natural killer (NK)-susceptible targets and syngeneic targets. Such killing was not inhibited by antibodies to H-2 or Lyt-2. In contrast, cultured mature Lyt-2+ L3T4- T cells from normal LN, thymus, or lpr LN were cytolytic only toward allogeneic targets and were dependent on Lyt-2 expression and H-2 recognition. The similarities of cultured Lyt-2- L3T4- T cells to NK and lymphokine-activated killer cells are discussed.