The recently discovered B6.C-H-2bm12 (bm12) histocompatibility mutant bears a mutation of the I-A subregion of the H-2b haplotype. We have studied cytotoxic T lymphocytes (CTL) generated in secondary mixed lymphocyte culture (MLC) between the bm12 mutant and the strain of origin C57BL/6 (B6). In both directions, strong CTL responses were generated against lipopolysaccharide-stimulated, but not against concanavalin A-stimulated target cells. Studies of the specificity of the bm12 anti-B6 CTL response, using target cells from a panel of H-2 recombinants, showed that the CTL were directed against specificities of the I-Ab subregion. This conclusion was confirmed by the observation that the bm12 anti-B6 CTL could be specifically blocked by anti-Iab, but not by anti-Kb antisera. EL4 tumor cells, which express Kb and Db but not Iab antigens, were not lysed by the CTL. The combined data confirm that bm12 is an I-A subregion mutant and indicate that the I-A subregion antigens, recognized by antibody and those recognized by CTL, are probably present on the same molecules. The CTL effector cells generated in secondary MLC across the I-Ab mutant difference had the surface phenotype Lyt 1- 2+.