In vitro culture of normal lymphohemopoietic cells is a complex event leading to the generation of a diverse group of effector cells. When murine spleen cells are cultured by themselves in fetal calf serum containing medium for up to 9 days, at least 3 distinct subpopulations of cytotoxic effectors can be distinguished on the basis of tumor target cell preference and expression of cell surface alloantigens. Fresh spleen cell suspensions contain 2 distinct NK cell types: NK-1.2+ NKA cells, which preferentially lyse lymphoma targets, and NK-1.2- NKB cells, which lyse nonlymphoma, mainly solid tumor targets. These NK cells account for all the cytolytic activity on day 0 of culture. NK-1.2+ NKA cells are very labile, and greater than or equal to 80% of their activity is lost within 48 hr of culture. By contrast, the activity of NKB cells increases in culture, up to 4-fold by day 6, and solid tumor target cells resistant to lysis by fresh spleen cells now become susceptible. In addition, an NK-1.2-, H-2+, Thy-1.2+, Ly-1.2+, Ly-2.2+ cytotoxic effector cell arises in culture from, or is dependent upon the presence of, NK-1.2-, Thy-1.2+ cells. This spontaneously arising "Tc" has a peak of activity between day 3 and 6 of culture, and kills a variety of lymphohemopoietic tumor cell types.