The D region of the H-2 d haplotype contains five class I genes: H-2D d, D2 d, D3 d, D4 d and H-2L d. Although previous studies have suggested the presence of D-end encoded class I molecules in addition to H-2D d and H-2L d, segregation of genes encoding such molecules has not been demonstrated. In this report we have used cytotoxic T lymphocytes (CTL) to examine the D region of the H-2 d haplotype for the presence of additional class I molecules. CTL generated in (C3H x B6.K1)F 1 (KkD k, KbD b) mice against the hybrid class I gene product Q10d/L a expressed on L cells crossreact with H-2L a but not H-2D d molecules, as determined by lysis of transfected cells expressing H-2L d but not H-2D d. Although H-2La-specific monoclonal antibodies (mAb) completely inhibit H-2La-specific CTL from killing B10.A(3R) (KbDdLd) target cells, only partial inhibition of anti-Q10 CTL-mediated lysis was observed, suggesting the presence of an additional D-end molecule as a target for these latter CTL. To identify the region containing the gene encoding the Q10 cross-reactive molecule, we show that anti-Q 10 CTL lyse target cells from a D-region recombinant strain B10.RQDB, which has H-2D d, D2 d, DS, D4 d, and H-2D b but not the H-2L d H-2 d, and H-2L d (including D2 d, DS, and D4d), lacks this anti-Q10 CTL target molecule. Together, these data demonstrate that a class I gene mapping between H-2D d and H-2L d encodes an antigen recognozed by anti-Q10 CTL. A likely candidate for this gene is D2 d, D3 d, or D4 d.