Two long-term human cytolytic T lymphocyte (CTL) lines (VE and JR), whose cytolytic activity was dependent upon both irradiated JY cells (the stimulating alloantigen) and T-cell growth factor, were established. These lines were monitored in culture for 6-8 months. Both lines were specific for HLA-A or B antigens or both and the JR line was allospecific for HLA-B7. These CTL lines killed specific target cells at an effector-to-target ratio of 0.4 (VE) or 0.08 (JR). All of the cells, which grow in suspension, rosetted with sheep erythrocytes and reacted with an antiserum specific for human T cells. The CTL line VE was used to raise rabbit antisera that immunoprecipitated two specific polypeptides (78,000 and 33,000 daltons) from labeled membranes of these CTL lines.