The fine specificity of alloimmune cytotoxic T lymphocytes (CTL) was investigated in CTL responses across the smallest known H-2 differences, those based on mutation at a single H-2 locus. CTL were generated in all possible mixed lymphocyte culture (MLC) combinations among seven H-2Kb mutants and the mouse strain of origin, C57BL/6 (B6-H-2b). CTL were also generated in all F1 hybrid responder/homozygous stimulator-cell combinations among four Kb mutants and B6-H-2b. CTL activity was measured in cell-mediated lympholysis (CML) against target cells from all Kb mutants and B6-H-2b. Cross-reactivity against targets other than the MLC stimulator was extensive and led to the distinction of 64 CML target determinants. Two Kb mutants (B6-H-2bm6 and B6.C-H-2bm9) showed identical typing for all 64 CML determinants. CML reactions after MLC between these two haplotypes were mutually negative. The mutants B6-H-2bm3 and B6.C-H-2bm11 showed identical typing for 47 of the 64 determinants. Their close relationship is in agreement with the finding that H-2bm3 anti-H-2bm11 CTL were the only ones that exclusively lysed target cells of stimulator-cell genotype. On the basis of CML typing, the sequence of relatedness of the mutants with H-2b is as follows: bm6/bm9-bm10-bm3-bm1-bm11, bm6/bm9 being the closest to, and bm11 the most distant from H-2b. The extensive cross-reactivity of alloimmune CTL appears to reflect immunogenetic complexity rather than lack of specificity. Comparison with other reports supports the notion that the system of Kb CML determinants, recognized by alloimmune CTL, is at least partially overlapping with the H-2Kb specificity repertoire involved in the associative T cell recognition of virus-infected cells.