Cytotoxic T-cells were derived from the peritoneal cavity of a C57BL/6 mouse immunized with BALB/c sarcoma Meth A and from the spleens of BALB/c x C57BL/6 F1 (hereafter called CB6F1) mice immunized with BALB/c leukemia RL male 1. The cells were cultured in interleukin 2 and cloned by limiting dilution, and their specificity was determined by direct tests and competitive inhibition assays. C57BL/6 anti-Meth A effector cells recognized H-2Dd determinants. CB6F1 anti-RL male 1 effector cells recognized a unique cell surface antigen of leukemia RL male 1. The specificity was maintained in long-term culture. The cell surface phenotype of the cloned effector cell lines as determined by absorption analysis was Thy-1.2+, Lyt-1.2+, 2.2+, and 3.2+. Cytotoxicity was blocked at the target cell level by antisera against H-2Dd, but not H-2Dk or H-2b, and at the effector cell level by antisera against Lyt-2.2 and 3.2, but not Lyt-1.2, Ly-5.1 or Thy-1.2.