The study of antigen processing and presentation by human antigen presenting cells (APC) has been limited by difficulties of producing and maintaining human T-cell clones. Murine T-cell hybridomas have advantages for detecting specific peptide-MHC complexes on APC. Human antigen-specific immortalized T-cell lines have not been successfully produced. We report and validate the use of transgenic mice with human MHC genes for HLA-A2, DR1 and DR4 to produce murine T-cell hybridomas that are restricted to human HLA alleles and respond to human macrophages, dendritic cells (DC), and B-cell lines. Hybridomas restricted by human MHC-I and -II specific for influenza matrix protein, tetanus toxoid, diphtheria antigen CRM(197), and various M. tuberculosis antigens were produced. Epitope specificity was determined for several hybridomas. T hybridomas recognized peptide-MHC complexes on fixed APC for analysis of kinetics or susceptibility to inhibitors of antigen processing. T hybridomas restricted by human MHC represent convenient and powerful tools for the study of antigen processing by human APC.