Peripheral blood lymphocytes (PBL) cultured in interleukin 2 (IL 2)-containing medium in conventional tissue culture develop the ability to lyse fresh tumor cells; such cells are referred to as lymphokine-activated killer (LAK) cells. LAK activity peaks by day 5 of culture and declines rapidly thereafter. We studied culture conditions and signals that allow for long-term culture and expansion of cells with LAK activity. By culturing cells at relatively low densities and regularly replenishing medium and recombinant IL 2 (r-IL 2), LAK function is significantly higher as compared with short-term cultures, and remains present for at least 21 days while cell numbers undergo an average 100-fold expansion. By activating these cultures with anti-CD3 (OKT3) monoclonal antibody and r-IL 2, an approximately 1000-fold expansion in the cell number is obtained with maintenance of comparable levels of LAK activity. The exogenous addition of beta interleukin 1 (beta-IL 1), interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma) can augment the lytic activity of cell populations expanded by anti-CD3 plus r-IL 2. These approaches may enable the in vitro generation from individual donors of much greater numbers of LAK cells for adoptive immunotherapy than can now be obtained with the 3 to 5 day in vitro culture systems.