Human allospecific cytotoxic T lymphocyte lines were established by weekly stimulation of peripheral blood lymphocytes with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines in the presence of interleukin 2. The cytotoxic T lymphocyte lines were stimulated by either JY (which expresses HLA-A,B and -DR) or Daudi (which expresses HLA-DR but not -A,B antigens). Specificity of the effector cell lines was determined by antibody blocking and by patterns of cytolysis against a panel of target cell lines. The phenotype of the effector cells was identified by both negative (antibody and complement lysis) and positive (fluorescence-activated cell sorter) selection. The anti-JY lines only recognized targets bearing HLA-A2, B7 antigens and were composed exclusively of OKT4-8+ effector cells. The anti-Daudi lines, on the other hand, specifically recognized targets bearing DR6 and eventually contained only OKT4+8- cells. Thus, stimulation by Daudi cells can result in the generation of OKT4+ CTL lines that are allospecific for DR6 antigen.