A method for infection of lymphocytes with Moloney(Abelson) murine leukemia virus [M(A)-MuLV] is described. Only lymphoblasts obtained after stimulation of normal spleen cells by the B cell mitogen lipopolysaccharide (LPS) were satisfactory targets for virus-specific, secondary cytotoxic T lymphocytes (CTL), whereas spleen cells stimulated by the T cell mitogen concanavalin A were not. The secondary CTL response against M(A)-MuLV could be efficiently measured using M(A)-MulV-infected LPS blasts as stimulating cells for secondary in vitro restimulation and as target cells for virus-specific destruction. Cold target inhibition demonstrated virus specificity of CTL. The T cell character of the cytotoxic cells was demonstrated by their sensitivity to anti-Thy-1.2 treatment. Using syngeneic virus-infected LPS blasts as target and stimulator, CTL responses were measured with effector cells from C57BL mice of the H-2b haplotype and of recombinant haplotypes sharing either K or D alleles with H-2b. In analogy with previous studies on Moloney virus-specific CTL, it was observed that C57BL/6 (H-2b) effector cells predominantly lysed Db-compatible, virus-infected target cells; B10.A(5R), (KbDd) effector cells showed a poor CTL response against syngeneic, virus-infected target cells. The combined findings indicate the existence of an Ir gene in the H-2D region regulating the CTL response against Moloney leukemia virus.