Cytotoxic T lymphocytes (CTL) were generated in secondary in vitro cultures against H-2-identical allogeneic stimulators. In some strain combinations, such as C57BL/10 (B10) responding to C3H.SW, cytolysis was restricted to target cells bearing the H-2 antigens of the immunizing strain. However, B10.BR effectors sensitized to C3H, AKR, or CBA efficiently lysed targets of several H-2 haplotypes, including B10 targets congenic with the B10.BR responders. The activity of these CTL was totally abrogated by anti-Thy 1.2 plus complement. Lysis of 51Cr-labeled B10 targets by B10.BR anti-AKR CTL was inhibited by unlabeled B10 and AKR targets equally well, thus showing that the clones killing B10 are included in the AKR-specific clones. In addition, both B10 and B10.D2 stimulator cells were effective in the in vitro boosting of the B10.BR anti-AKR cytotoxic response. Mapping experiments using B10, B10.A, B10.A(2R), B10.A(5R), C57BL/6, and B6-Tla(a) mice indicated that the locus relevant to these phenomena maps to the right of H-2D. Since both Con A and LPS blast targets were susceptible to the activity of the nonrestricted CTL, antigens encoded by this locus are not unique to the T or B cell lineage.