Protective immunity against mycobacteria is dependent on antigen/MHC class II specific, CD4+ Th1 cells. HLA-DR3-restricted Th1 cells respond to a subset of mycobacterial antigens, including the immunodominant hsp65, and recognize a single epitope in hsp65, notably p1-20. Altered peptide ligands (APL) of p1-20 can inhibit p1-20/hsp65-induced proliferation of DR3-restricted T cells in an allele specific manner in vitro. In order to develop a preclinical model in which p1-20 APL can be tested in vivo in the context of HLA, we have used murine class II deficient, HLA transgenic (Ab0) mice, in which all CD4+ T cells are restricted by the tg HLA molecule. BCG-immunized DR3.Ab0 and DQ8.Ab0 mice both responded well to hsp65. Furthermore, DR3.Ab0 mice recognized precisely the same p1-20 epitope as DR3-restricted human T cells, whereas DQ8.Ab0 mice responded to a different set of hsp65 peptides. This shows that (i) the same immunodominant protein and peptide epitope are recognized by T cells from DR3.Ab0 mice and DR3+ humans and (ii) indicates the major role of HLA-polymorphism in controlling the human T cell response to mycobacterial antigens. Thus, HLA-transgenic, Ab0 mice provide a novel, preclinical model system to analyze APL and vaccines in the context of HLA polymorphism.