The alloantigens encoded by the four defined Qa-1 genotypes were characterized by cloned cytotoxic T lymphocyte (CTL) recognition. CTL clones specific for Qa-1(a)- and for Qa-1(b)-encoded antigens were generated. Examination of the reactivity of these clones with target cells from H-2(r) and H-2(f) strains provided the strongest evidence to date for the designation of the Qa-1(c) and Qa-1(d) genotypes, respectively, for these strains. Qa-1(c)-encoded antigens were recognized by most, but not all CTL clones that specifically lysed Qa-1(b) target cells, thus demonstrating that these antigens lack a Qa-1(b)-associated determinant. Similarly, Qa-1(d) encoded antigens were recognized by only half of the CTL clones that lysed Qa-1(a) target cells. In addition, one CTL clone that was cytogenic for Qa-1(b) and Qa-1(c) target cells demonstrated low affinity, cross-reactive recognition of a Qa-1(d) encoded antigen. The reactivity patterns of the monoclonal CTL defined five Qa-1 determinants. Qa-1(a), Qa-1(b) and Qa-1(d) each encode multiple determinants. Two Qa-1(d) encoded determinants probably reside on different molecular species. Finally, large numbers of CTL clones tested on panels of target cells indicated that the Qa-1(a) strains expressed indistinguishable Qa-1.1 antigens and the Qa-1(b) strains expressed indistinguishable Qa-1.2 antigens. Therefore, additional polymorphism among these strains is improbable.