Cloned genes for human major histocompatibility complex (MHC) class I antigens were introduced by DNA-mediated gene transfer into a high-efficiency transfection recipient (HTR) cell line previously derived from mouse mastocytoma P815. Cell surface expression of HLA-A3, AW24 and CW3 gene products on P815 transfectants was demonstrated by radioimmune assay and by flow cytometry. The human MHC class I gene products were apparently expressed on P815 transfectants in a form recognized by human cytolytic T lymphocytes (CTL). Human CTL generated in unidirectional mixed lymphocyte culture against AW24+ donor lymphocytes clearly lysed P815-HLA-AW24+ transfectant target cells, but not untransfected P815(HTR) controls. Moreover, P815-HLA transfectants could stimulate in vitro a significant alloreactive human CTL response. Lysis of P815-HLA transfectant target cells by human CTL was inhibited by a monoclonal antibody directed against human MHC class I gene products. These mouse cell transfectants may be useful for the study of human T cell responses.