Cytotoxic clones consisting entirely of large, vacuolated granular lymphocytes (LGL) are produced with high frequency when individual purified mature Ly 2+ T cells are cultured at limit dilution in the presence of concanavalin A and irradiated spleen filler cells. Similar cells are produced in cultures of Ly 2- T cells but in lower proportion, the level of granular lymphocytes amongst the largely non-cytotoxic, Ly 2- L3T4+ product cells being only 20%. A proportion (15%) of LGL are also found in conventional mixed lymphocyte cultures. In the limit-dilution cultures of Ly 2+ T cells the LGL originate from the single precursor cell and not from the irradiated filler cells. They bear the markers expected of active cytotoxic T lymphocytes (Thy 1++; PNA++; Ly 1+; Ly 2++; L3T4-). Their DNA and chromosome complement appear normal. The granules within the cells are heterogeneous in form, and some resemble in ultrastructure those described in certain cytotoxic, suppressor and NK-cell lines. The granules do not show mast cell staining characteristics. The LGL are not phagocytic. Although LGL are associated with nonspecific cytotoxicity late in culture, the first granular cells appear early (day 3) and dominate in the clones at times when cytolysis is specific. LGL appear to be the normal morphological form of cytotoxic T lymphocytes when grown under these conditions.